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Yellow Rhododendron Dark Background is a painting by Sharon Freeman which was uploaded on June 23rd, 2013.
Watercolor rhododendron with colorful shadows and a wet in wet abstract background.
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Yellow Rhododendron Dark Background
Sharon Freeman
Painting - Watercolor On Paper
Watercolor rhododendron with colorful shadows and a wet in wet abstract background.
June 23rd, 2013
Viewed 552 Times - Last Visitor from Sunnyvale, CA on 07/19/2017 at 5:35 PM
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If you only care about trump attacking Syria because it could start a major war and not about the fact that syrians have been suffering for years at the hands of Assad to then having America constantly destabilize/adding more trauma and deaths and now in their darkest times will continue to suffer, then you're an asshole and you really dont care about anyone but yourself lmao
President Trump has launched 59 Tomahawk missiles, striking Syria. We have gotten further involved in another war in the Middle East. This action will only further complicate the troubles in the region. We will see just how as the reactions unfold.
So I'm not OLD old but I do want to point out that Bush was polling real fucking low before we all went to war in Iraq and that Thatcher was on the brink of irrelevance before the Falklands.
The fact that May's cabinet* is being militaristic and belligerent toward Spain and that POTUS 45 has launched 50 warheads into Syria is a political act to solidify authority.
In the coming months the UK and USA are going to have patriotism and glory-baiting shoved down our throats.
So remember these things:
1) War is hell, don't let your representatives condone it.
2) You can support the men and women in the armed forces as individuals without supporting the military-imperial business model.
3) Times of war are when leaders must be held the most accountable for their actions, not rallied behind for the greater good.
The UK Government has yet to issue any new statement regarding military action in Syria. This is not a good time to live in.
Peace is an open hand, not a Tomahawk warhead.
*EDIT: William Hague is a Tory Life Peer, former party leader, former First Secretary of State, and Former Secretary of State for Foreign and Commonwealth Affairs, among other titles. While not part of the Cabinet as claimed, he is still politically relevant.
If you are or know anyone who is at an airport:
Please protect yourself.
EDIT: Also, if you're a green card resident who is a citizen of or from IRAN, IRAQ, LIBYA, SOMALIA, SUDAN, SYRIA, OR YEMEN:
Please make sure you DO NOT LEAVE THE UNITED STATES UNDER ANY CIRCUMSTANCES. YOU WILL NOT BE PERMITTED TO RE-ENTER THE COUNTRY.
Mni Wiconi, Water is Life
Solidarity With Refugees
What will it take to get this message across? Spread this like wildfire please.
does anyone else have a sick feeling in their stomach right now? i honestly don't know how i feel about the situation in syria, but i know the incompetent half-sentient cheeto in the white house isn't equipped to handle a fucking war
cnbc article: [x]
Syrian kids deserve a chance to feel the childish joy of running out of their houses at the sound of an ice cream truck and buying ice cream
Pakistani kids deserve the chance to go to an amusement park and feel the rush of the wind in their face as they try the biggest rollercoaster in the park
Afghan kids deserve a day to sleep in and wake up lazily to the warm glow of the sun and the sound of happy voices coming from downstairs
A young Iraqi girl deserves the chance to discover the amazing world of cake decorating and realize that she was born to decorate incredibly stunning cakes
A boy from Uganda deserves to fulfill his dream of becoming a singer because his whole family and all his neighbours always praise his beautiful voice
A little Somali girl deserves to know all the different art mediums that exist so she can explore her God given talents as an artist and mesmerize everyone with her work
A brown man enslaved in Kuwait deserves to wake up early and surprise his wife with breakfast in bed and to hold her in his arms
A little boy in North Korea deserves to feel the nervous excitement as he gets on a plane to fulfill his dream of traveling the world
A Palestinian girl deserves her chance to stun the world with her superior acting skills, a surefire Oscar winner if the world ever knew it
A Kashmiri boy deserves a chance to delve into the world of literature and be the greatest poet we ever knew
A Native girl in Canada deserves to find out she's an amazing swimmer and fulfill her dream of one day competing in the Olympics
A Sudani boy deserves to feel the excitement of seeing a magic trick for the first time and then putting on his own little magic show for the neighborhood
An Egyptian girl deserves to find out she can make the best blueberry muffins you ever tasted and she opens her own bakery to discover she's a talented businesswoman as well
A black boy in America deserves the chance to graduate from his dream law school and become the best humanitarian lawyer to practice
A Nigerian boy deserves the chance to wake up at dawn and go on a beach trip with his friends so they can catch the sunrise and stare in awe at the explosion of colours against the sky
A Yemeni girl deserves to feel the exhaustion after spending an entire day doing dares with her friends and she goes to sleep with a smile on her face
If they were born on this planet, they were meant to be here. Everyone deserves a chance at a life. They deserve to know peace.
The daily struggles of the children around the world should include arguing with their parents about not wearing a sweater because it's not that cold out, not making sure they live to see another day.
Everyone deserves a chance at life.
Do what you can to save humanity. Change starts with you.
"Never Forget" should mean that, not arming Saudi Arabia and applauding U.S. imperialism's continued bombing of the middle east.
This attack is not helping Syrians in any way shape or form?? Some of you actually think trump is doing Syrians a favor like how ugly do you have to be to believe that??? Idgaf what Trumps intention with the airstrike is, INNOCENT people WILL die. Donald Trump hiding behind Asaad's regime is everything that is wrong with this. Innocent people will die and it will be HIS fault. He isnt doing ANYONE a favor and you're disgusting if you think this is his way of protecting Syrians from Assad's regimes and ISIS.

subset: commoncrawl


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Pôrodné asistentky
Pozrite si súvisiace témy
QD Memes - QD Nurses - NCLEX Flash Cards to Help You on Your Next Exam
Fetal Station #OB #Nursing ( I am pinning these from my blog lulabelleismyshihtzu.tumblr.com )
This was always confusing in school. I like how it is presented here, so I thought I'd share the wealth...
These are great! Left the blog info, not taking credit for these, but sure wish I had thought of doing this. StagesStages Of Labor #OB #Nursing ( I am pinning these from my blog lulabelleismyshihtzu.tumblr.com )
Find out more about Natural Birthing Here ==>> www.HypnobirthingRocks.com
Fetal heart monitor interpretation. ....nursing So imagine having a frame like this for when the character is about to die or enter a heart attack, etc., for the comic. It'd be a great use of incorporating semiotics, as well as breaking the social norm of comic strip formats!
I'm 5 months pregnant right now, and STILL have no fucking idea what any of this means. Baby Mahar is somewhere in between my bladder and my last nerve.
MG SO4 TOXICITY Signs and Symptoms: BURP
Postpartum Assessment: "BUBBLE-HE"
50 Nursing Mnemonics and Acronyms (Nursing Assessment): http://www.nursebuff.com/nursing-assessment-mnemonics/


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The mom of one wowed in a swimsuit while carrying her nephew as she enjoyed some pool time.
Milian, who has a daughter with singer The Dream, showed off her busty frontier as she effortlessly slayed in the swimwear.
EnterGhana.com | Credit: Misspetitenaijablog.com | Christina Milian Puts On A Busty Display In Swimsuit
Have a say. What do you think? Drop comments!


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Time in Norway: 4:45 AM
Make a search for products & suppliers, articles & news.
High definition cameras from Norwegian high-tech company Kongsberg Maritime were used in a recent expedition of China's manned deep-sea submersible Jiaolong to the Mariana Trench in the western Pacific Ocean.
High definition cameras from Norwegian high-tech company Kongsberg Maritime were used in a recent expedition of China's manned deep-sea submersible Jiaolong to the Mariana Trench in the western Pacific Ocean.
The Jiaolong had been equipped with two ultra deep water versions of Kongsberg Maritime OE14-502D High Definition Cameras along with an ultra deep OE14-208 Digital Stills Camera and two OE14-364 Compact Fixed Colour Cameras. The Jiaolong made a total of six dives altogether, reaching a depth of 7062 meters on its final dive. This surpassed a national record depth of 7020 meters reached just a few days previously.
While at the ocean bottom, scientists captured high quality video, conducted geological surveys, placed markers and gathered samples of organisms living on the ocean bed.
In a company press release, Kongsberg Maritime says the HD video footage is the deepest recorded using KONGSBERG HD cameras. This, along with numerous past deep water marine research applications around the globe where KONGSBERG cameras have been used in depths in excess of 8000 meters, demonstrates KONGSBERG's ability to manufacture the most rugged harsh environment underwater cameras available.
Eimskip has strengthened its position in worldwide logistics services by acquiring 75% of the freight forwarding company SHIP-LOG.
For Sale Assets from the former BMV LAKSEVÄG YARD in Bergen, Norway! The entire shipbuilding and fabricating facility is closing and more than 500 lots is on sale.
We are proud to announce that Servogear has been chosen for the delivery of Servogear...
The associated activities will be managed by TechnipFMC Process Technology's center in Houston,
The Norwegian Research Council has granted 10 million...
Oglaend System's headquarters will remain in Klepp,
The Harvey Deep Sea Vessel is a Multi-Purpose DPII Construction,
The Norwegian Radiation Protection Authority has become aware that stories about an ongoing incident involving a "meltdown" at the Norwegian Institute for Energy Technology (IFE) reactor situated in Halden are currently ci...
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Normal ice cubes from the freezer? Anyone can have them! Our fruity ice lollies add a very special refreshing touch to your summer drink: frozen pieces of fruit on skewers not only look fancy, they also keep your drinks cool on hot days and ensure an extra boost of vitamins. Here we show you how you can make your own fruity ice lollies for your drinks.
FroYo Bites: hip summer refreshment
We love FroYo Bites! These tiny sweet treats from the US are THE trend of the summer and a fruity alternative to ice cream and other sweet desserts. These hip snacks, made from frozen fruit coated in yoghurt, are quick to prepare and are the ideal snack when you are on the go or enjoying a sociable get-together.
Fruity couscous salad
Fruit alert in salad! Couscous is traditionally a popular savoury dish but this fresh and fruity version is our favourite way to enjoy the healthy culinary delight.
Healthy summer snack: Fruit salad in cones
Summer is just around the corner, which means garden parties, open-air children's birthday parties and other fun events. Sweet and fruity snacks are a must for every buffet table. Our recommendation of fruit salad in cones – decorated with raspberry sauce and crunchy nuts – is guaranteed to be a hit!
Pineapple sesame snack: perfect for picnics
Is there anything nicer than a picnic in the great outdoors with your loved ones and a few tasty snacks? Today we have a pineapple sesame snack for you that will be the perfect addition to your picnic. The sweetness of the fruit, combined with a sweet and nutty sesame and honey coating, makes for a very special delicacy and is guaranteed to be a hit with young and old. It doubles the enjoyment of a day in the great outdoors.
A boost of energy when you are drained: banana café frappé
You got to bed later than you planned again last night – now you are exhausted and finding it difficult to concentrate. You need a quick energy boost to wake you up. Our banana café frappé, an exotic change from a normal coffee, is just the answer. It includes an extra portion of vitamins to get body and soul going again.
Colorful fruity wraps
Fancy the taste of summer all year round? It's easy with our colorful fruity wraps. They are super-quick to make and the ideal snack when you are on the go. And the best thing is: everyone can roll up their favourite fruit and conjure up their own personalised wrap.
Roasted vegetables with fruit
Summer has not begun yet, and a barbecue outside is probably not much fun given the current temperatures. But we can bring the ultimate summer barbecue feeling direct to your kitchen: our fruity roasted vegetables are ready in no time at all and the result is a summery fruity delicacy and no cold hands or feet.
Vitamin shake
Looking for some delicious and revitalising refreshment? Then we have exactly the right thing for you! Whether you want to boost your vitamin C levels or simply feel like some fruity refreshment, our vitamin shake is always a good idea. The slight taste of turmeric also ensures a special type of taste experience.
Waldorf salad with grapes
Now an international favourite, the Waldorf salad has its roots in America: Oscar Tschirky, a waiter at the exclusive Waldorf Hotel in New York, created the original recipe in the late 19th century. Here we give this classic dish a modern twist and refine the Waldorf salad with crunchy walnuts and juicy grapes.


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AMAZING CUT PRICE OFFER ON VALTRA'S COMPACT A93!February 2, 2016
KICK START SPRING 2016 with a new tractor! Buy a NEW VALTRA A93 from Hamilton Brothers before April 5th at LAST YEAR'S PRICE!
The Agricultural sales staff at Hamilton Brothers have been beaming from ear to ear following the success of our first full year as Valtra agents and, to celebrate, we received a batch of A93 tractors on special offer. This means that our loyal customers can now purchase this top selling tractor at last year's price!
Praised as Scandinavia's most popular tractor, the A93 is the ideal stockman machine for our Scottish crop and arable farmers. Both strong and agile, the A93 epitomises the no nonsense reliability of the Valtra brand and, with an impressive resume of key features to boot, it is no surprise that this terrific tractor is a top seller.
Key features of Scandinavia's Top Selling Tractor:
- 3 Litre AGCO Powered Siso Engine
- 75Kw 100hp 370Nm
- Excellent Front Loading Tractor with Heavy Duty Front Axle
- Excellent Weight Distribution for a Wide Range of Applications
- Long Wheelbase for Enhanced Stability
- Superb Manoeuvrability in Farm Yards & Headlands
- Auto Control
- Electro Hydraulic Clutchless Shuttle
- Comfort, Economy, Versatility
Hurry, Buy Before 5th April for Best Savings!
Hurry, the end of the tax year is fast approaching! If you want to reap the full benefit of this fantastic deal, then purchase your A93 before April 5th to offset any potential implications and save even more! At Hamilton Brothers our Financial Conduct Authority regulated advisers are always on hand to help and find a deal that best suits your needs.
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- Drive the Latest JCB Agriculture Range at our Hamilton Ross Group JCB 2017 Roadshow!
- Valtra's New 4th Generation A Series Celebrates UK Launch!
- The Countdown is on for The Royal Highland Show!


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My family and I stayed at this hotel for 4 nights. The hotel is close to the highway with several great restaurants, a Target and shops an exit away. The staff at the hotel were very accommodating and could not be nicer. We were allowed...More
Getting you more information on this room
Getting you more information on this room
"Request away from highway for very Quiet room but then view may bot be the best."
"Have a vehicle, or use Uber / Lyft, a bit out the way from downtown / cabs are $35 or more"


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ENDS TONIGHT! Up to 60% OFF Cards, Invites, Aprons, Stickers, Mugs and More! | + 20% OFF SITEWIDE | Use Code: JULYXMAS2017 | See Details
Rustic Wood and Lace Wedding Invitation
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60% off with code JULYXMAS2017
12.7 cm x 17.8 cmMore (8)
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- 17.5 pt thickness / 120 lb weight
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Size: 12.7 cm x 17.8 cm Invitation Card
Make custom invitations and announcements for every special occasion! Add photos and text to both sides of this flat card for no extra cost. Choose from twelve unique paper types, two printing options and six shape options to design a card that's perfect for you.
- 12.7 cm x 17.8 cm (5" x 7"), portrait, or 17.8 cm x 12.7 cm
(7" x 5"), landscape. Additional sizes available.
- High quality, full-colour, full-bleed printing on both sides.
- Standard white envelope included.
- 12 unique paper types and colours. Learn more.
- Discover what makes Zazzle Invitations unique. Learn more
- Designer Tip: To ensure the highest quality print, please note this product's customisable design area measures 12.7 cm x 17.8 cm
(5" x 7"). For best results please add 0.16 cm (1/16") bleed.
Printing Process: Standard Printing
Our printing process produces a full colour spectrum. Using liquid inks, our high quality printing rivals the quality of flat printing, ensuring your images and designs are beautiful and vibrant.
Paper Type: Signature Collection: Matte
The most popular paper choice, our Matte is ideal for invitations, RSVP cards, and other paper-based party essentials. Matte's eggshell texture is soft to the touch with a smooth finish that provides the perfect backdrop for your chosen designs.
- 175 gsm, 0.43 mm thickness.
- Soft, eggshell texture with smooth finish.
- Made in the USA.
Will be made by
Zazzle Invitations
San Jose, CA
About This Design
Rustic Wood and Lace Wedding Invitation
Rustic modern outdoors wedding invitation design with elegant lace elements at the top and bottom on a printed faux wood texture background. An elegant and stylish rustic design, perfect for outdoor or vintage themed weddings! Click the CUSTOMIZE IT button to customise fonts, move text around and create your own unique one-of-a-kind invitation design.
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text: Definition Edit Message externals are " [i]nformation outside of the message text, such as the header, trailer, etc.[1] " References Edit ↑ CNSSI 4009. Retrieved from "http://itlaw.wikia.com/wiki/Message_externals?oldid=172604" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Definition Data E-mail Add category Cancel Save


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WATCH: Kingswood Get All Horned Up W/ Destiny's Child For 'Like A Version'
After the recent cover of 'Hold Up' by the very good folks in Holy Holy, we have been once-again blessed by Queen Bey, with Kingswood stepping into the Triple J studios this morning to serve up an absolutely mammoth, knock-ya-socks-off rendition of 'Say My Name by Destiny's Child.
How the shit did we ever get so blessed as to have two Bey-heavy tracks in the space of a month? We are surely not worthy.
The Kingswood boys brought in a whole crew of friends to help them turn the already-huge banger into something absolutely monumental. I mean, they have a horn sections for God's sake. A HORN SECTION.
I'm calling it – this is going to be the 'Like A Version' of the year. Keep this one in your back pocket for when you're voting in the Hottest 100 later on this year.
Clock your peepers on this one, fam. It's bonkers good.
All covers to be Beyoncé from here on out, please and thank you.
Photo: Triple J / Facebook.


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The Quiet Power of Indicators: Measuring Governance, Corruption, and Rule of Law by Cambridge University Press (Paperback, 2015)
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About this product
- DescriptionThis highly accessible book investigates the rankings that increasingly influence perceptions of countries' governance and civil rights.
- Author BiographySally Engle Merry is Silver Professor of Anthropology at New York University. She is the author and editor of nine books, including the J. Willard Hurst Prize-winning Colonizing Hawai'i: The Cultural Power of Law (2000). Her most recent book is Governance by Indicators (2012, co-edited with Kevin Davis, Angelina Fisher and Benedict Kingsbury). Kevin E. Davis is Beller Family Professor of Business Law and Vice Dean at New York University School of Law. He has written extensively about anti-corruption law as well as the relationship between law and economic development in such journals as the American Journal of Comparative Law, the Law and Society Review, and the New York University Law Review. Benedict Kingsbury is Murry and Ida Becker Professor of Law and director of the Institute for International Law and Justice at New York University School of Law. In addition to being co-Editor-in-Chief of the American Journal of International Law he is the editor and co-editor of over fifteen books, including International Financial Institutions and Global Legal Governance (2011).
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- PublisherCambridge University Press
- Date of Publication19/05/2015
- Language(s)English
- FormatPaperback
- GenreLaw: General & Reference
- Series TitleCambridge Studies in Law and Society
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- Country of PublicationUnited Kingdom
- ImprintCambridge University Press
- Content Note8 b/w illus. 9 tables
- Weight500 g
- Width152 mm
- Height228 mm
- Spine19 mm
- Pagination369
- Edited byAngelina Fisher,Benedict Kingsbury,Kevin E. Davis,Sally Engle Merry
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Ronald Gaiser | Birmingham Business Lawyer
Ronald Otto Gaiser
Don't Panic, Call for Free Initial Consultation
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Accident & Injury, Car Accident, Personal Injury, Medical Malpractice, Class ActionBirmingham, AL 35213 (Jefferson County)
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Asbestos & Mesothelioma, Natural Resources, LitigationBirmingham, AL 35205 (Jefferson County)
Construction, Products Liability, Sexual Harassment, Landlord-Tenant, InsuranceBirmingham, AL 35209 (Jefferson County)
Criminal, Felony, Misdemeanor, DUI-DWI, TrafficBirmingham, AL 35209 (Jefferson County)
Accident & Injury, Employment, Wrongful DeathBirmingham, AL 35203 (Jefferson County)
Employment Discrimination, Workers' Compensation, Wrongful Termination, Sexual Harassment, Personal InjuryBirmingham, AL 35236 (Jefferson County)
Bankruptcy & Debt, Personal Injury, Divorce & Family Law, EstateBirmingham, AL 35203 (Jefferson County)
Criminal, Accident & Injury, Business, Divorce & Family LawBirmingham, AL 35203 (Jefferson County)
Divorce & Family Law, CriminalBirmingham, AL 35243 (Jefferson County)
Divorce, Family Law, Child Custody, Felony, MilitaryHomewood, AL 35209 (Jefferson County)
Employment, Personal Injury, Sexual Harassment, Accident & Injury, Wrongful TerminationBirmingham, AL 35236 (Jefferson County)
I and my firm are qualified to represent potential litigants in all of the courts of Alabama, including the United States, Northern and other District Courts of Alabama, District Court of the District of Columbia, the Eleventh Circuit Court of Appeals, and the Supreme Court of the United States. I and my firm for the most part represent plaintiffs in almost every area of trial law, including personal injury, foreclosures, breaches of contracts, fraud and misrepresentation, ERISA law and most any other major areas of law and have done so for many years.
Lawyer.com Member Questionnaire
How did you build a successful practice?
Prior to becoming an attorney, I was a very experienced business man and salesman, which knowledge was gained from my prior 20 year career as the State of Alabama manager and agent of Provident Mutual Life Insurance Company and National Life Insurance Company of Vermont. In this regard I was also a Life Member of the Million Dollar Round Table and the Five-Million Dollar Forum. Also following completion of law school I was appointed Professor of Insurance, where I served for 5-years as Professor of Insurance.ed Registered Investment Advisor. Also, upon graduating from law school magnum cum lade I was appointed by the Dean of my law school as Professor of insurance law, which served for the next several years following my attaining my law license. My partner and I have reached out across much of the state of Alabama to persons of all races and national origins, including accepting representation and often partnering with local lawyers for the past 30-years. several years many who have becoming good friends and partners in complex litigation, which we have specialized in, one of whom was a very well known black attorney J.L. Chestnut, Jr, who fame in Alabama included his having representing Martin Luther King and other Civil Rights leaders during the civil rights movement several years ago. Later, upon meeting J.L. and his family my firm joined together with J.L.'s to to represent many black residents who had been schemed against by having been sold bogus insurance products, and so-called investments and retirement plans as well as other areas of fraud and misconduct toward people of color, by unscrupulous venders and hustlers. I would merely say that as I told my former my law students time after time that a lawyer must follow the "golden rule," which commands each of us to follow the Golden Rule to "do unto others as they would do unto you," and that anything less is un acceptable. This biblical command requires a lawyer to always put his client's interests ahead of himself...and "always means always.'"
What should clients look for in a lawyer?
It starts with the gravity of the individual's problem. For example if the person had not paid their taxes for several years and the IRS filed criminal action against them they best find both an accountant and an attorney well versed in such claims and knowledgeable of the United States Tax Code...which would would not be me. Likewise, those who find their lawyer from the ad on a billboard are not likely to that the attorney will be personally interested in them, in that even if he or she was the lawyer will unlikely have much time to act for them because he or she will have to be on the phone all of the time.
How important is local knowledge to the success of your cases?
I married a young lady who was born and grew up in the very areas of Homewood and Mountain Brook, Ala. where, we have lived for the past 30-years and where we have attended church together, and raised our two children, and now are helping raise four grandchildren, that with the exception of my last three years of college at the University of Arizona, following my honorable discharge from having served on a General Staff in the United States Army' that my wife and have lived in the same community of Homewood and Mountain Brook Alabama
What information can you provide in a free phone consultation?
It depends on what the potential client's knowledge of the origin and complexity of the potential problem they needed help on. For example if the person's problem was that they have just been charged with a murder, you would want to tell them to keep their mouth shut until you an opportunity to look over the facts , interview your client and potential witnesses, etc. etc. While should a party calling for advice want's advice as to whether they should pay their federal taxes on time, the advice would be rather simple and straight forward..."of course, go to a competent tax preparer...you don't need a lawyer.
What differentiates you from other lawyers in your community?
To whom much is given, much is expected, thus every day and every minute I push my self to do the very best I can, as the Master has said ,"To whom much is given, much is expected." and whether I am praying for Gods's grace for a client charged with murder, whom I believe is innocent or I am playing in the British Amateur at Saint Andrews golf club in Scotland, or merely hitting a soft wedge on the practice tee, I have always pushed my self to hit the shot as very best as I can.
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Both my wife and I have always been involved in our community,but not nearly as much as our church Dawson Memorial Baptist's activities which is right now engaged in a $5 million dollar mission program to spread the gospel of Jesus Christ, including helping build a hospital in a remote area of Africa
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Was apparently a goldminer in the 1890's.
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Thanks Andrew for finding these records on the internet! That's great!
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Thanks for digging up the gold and patent info. If you find anything else, please let me know.


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We describe the use of a unique DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5-hydroxymethylcytosine (5hmC) at low-occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follow the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the monomethylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.
The dynamic epigenomic landscape of the mammalian cell, including chromatin states and DNA modifications, is set forth by past cellular events, and it affects the developmental trajectory of the cell by regulating the genome-wide expression. DNA modifications such as 5-methylcytosine (5mC) are laid onto the genome by various DNA methyltransferases (e.g., DNMT1, DNMT3A, and DNMT3B) and undergo cycles of erasure and re-establishment at both genomic and regional levels. The unique methylomes are largely maintained in differentiated cell types, making them a key feature in understanding the differentiation potential of the cell. Recently, it was shown that 5-hydroxymethylcytosine (5hmC) is an oxidation product of the TET1/2/3 and is enriched in neuronal tissues as well as in embryonic stem cells (ESCs) (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009). Successive oxidation of 5hmC by the TET enzymes produces 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), which are found at a lower abundance in the genome (He et al., 2011; Ito et al., 2011). It was then realized that conventional cytosine methylation profiling methods, including bisulfite sequencing, cannot distinguish between 5mC and 5hmC (Huang et al., 2010; Jin et al., 2010). Hence, dissection of the hydroxymethylome from the methylome has become a priority in understanding its role in the genome regulation circuitry.
A number of methods have been developed to investigate the genome-wide distribution of 5hmC and its dynamics. For example, based on the differential sensitivity of MspI and HpaII to modified cytosines, 5hmC/5mC/C levels can be quantitatively estimated at any CCGG sites (Kinney et al., 2011). Several genome-wide studies have used 5hmC-specific antibodies (hMeDIP) or chemical affinity tags to enrich 5hmC-containing DNA for sequencing (Song et al., 2011; Ficz et al., 2011; Pastor et al., 2011; Williams et al., 2011; Wu et al., 2011). Although limited by resolution, these studies yielded many insightful observations. Recently, two methods of base-resolution hydroxymethylome mapping were reported (Booth et al., 2012; Yu et al., 2012). Both methods utilized the differential readout of various modified cytosines after bisulfite conversion and sequencing. 5mC and 5hmC are read as cytosine after bisulfite sequencing, whereas further oxidized products of 5hmCs (5fC or 5caC) are read as thymine. One method (oxoBS-seq) achieved the selective chemical oxidation of 5hmC to 5fC by using potassium perruthenate (KRuO4) (Booth et al., 2012). The other method (Tetassisted bisulfite sequencing, TAB-seq) first blocked the existing 5hmC by glucosylation and then converted all genomic 5mC to 5caC by utilizing the in vitro enzymatic oxidation of TET1 (Yu et al., 2012). Both methods used traditional bisulfite conversion and sequencing to decipher methylome and hydroxymethylome. However, several technical hurdles may still persist. For example, DNA complexity and integrity are further compromised after the treatment which may hamper the amplification steps and the subsequent read mapping. In addition, sequencing depth for each cytosine must be considerably high to detect low-level 5hmCs (Yu et al., 2012).
Alternative methods, ideally with a facile application format, are needed to complement the existing approaches. In previous work we characterized a family of 5hmC-dependent restriction endonucleases (Wang et al., 2011). One member in the family, AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and C, and cleaves at a narrow range of distances away from the recognized modified cytosine. By mapping the cleaved ends, the exact 5hmC locations could be determined (Wang et al., 2011). Here we report the genome-wide high-resolution hydroxymethylome of the mouse E14 ESCs using AbaSI-coupled sequencing (Aba-seq).
DNA modification-dependent restriction endonuclease AbaSI recognizes 5gmC with high specificity and generates double-stranded breaks at the 3′ side of the binding sites (Wang et al., 2011). AbaSI does not digest 5mC or C under the conditions used (Figure S1A). The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority 11–13 nucleotides away in the top strand and 9–10 nucleotides away in the bottom strand (Wang et al., 2011). The recognition and cleavage sites of AbaSI are shown in Figure 1A. AbaSI prefers sites with two cytosines positioned symmetrically around the cleavage site: one is 5gmC and the other can be modified or not (Figure 1A). AbaSI cleaves with a lower efficiency when only one 5gmC is present (Wang et al., 2011). Conversion of genomic 5hmC to 5gmC was completed by using the T4 β-glucosyltransferase (Terragni et al., 2012) (Figure 1B). The AbaSI-digested DNA was first ligated to a biotinylated DNA adaptor P1b containing 2-base randomized 3′-overhang (Figure 1B). After fragmentation and end polishing, bead-captured DNA was ligated to the second adaptor P2 (Figure 1B). Sequencing was initiated from the P1b end, and the AbaSI cleavage sites were revealed after mapping the reads to the reference genome. We determined that as low as 50 ng of E14 genomic DNA is sufficient for successful library construction (Figure S1B).
Putative 5hmC sites can be deduced by checking the presence of cytosine at expected distances (N13/N11,N12/N10,N11/N9) from either side of the mapped cleavage sites (Figure 1A). Based on the cytosine presence around the cleavage sites, we categorized them into groups (Figure 1C), e.g., 1CG1CH signifies one CG and one CH (H = A/C/T) at either side of the cleavage site respectively; similarly, 1CG1XX signifies one CG on one side and no cytosine on the other side. In groups with cytosines on both sides, we prioritized the 5hmC assignment in the CG context over the CH context. For example, 5hmC is assigned to the cytosine in the CG context in the 1CG1CH group. For over 82% of the cleavage sites, e.g., 1CG1CH, 1CG1XX, 1CH1XX, 5hmC can be assigned to a single cytosine with high confidence. There is assignment ambiguity to the exact cytosine base in categories such as 2CGs or 2CHs and accounts for 13% of all identified cleavage sites. These sites were included in our analysis as one 5hmC site. A representative sequence logo is shown in Figure 1D for the 1CG1CH sites, demonstrating little bias exists besides the recognized cytosines. There are only 5% of the mapped cleavage sites without suitable AbaSI recognition cytosines, in contrast to an expected 25% by chance (Figure S1C). With two technical replicates starting from AbaSI digestion, we obtained 242.0 (library R1) and 228.0 (library R2) million reads, of which 131.8 (54%) and 114.3 (50%) million reads were unambiguously mapped to the reference genome. Within mapped reads, 124.7 and 107.8 million 5hmC-induced reads were called, corresponding to 17.6 and 15.2 million 5hmC sites respectively (see Methods). There is a high degree of reproducibility between the two replicates with 12,229,723 unique 5hmC sites in common, which was used in the following analyses (Figure S1D). The mean and median of the combined sequencing coverage for these common 5hmC sites are 12.3X and 9X respectively (Figure S1E). In addition, within each library, sequencing coverage for the common 5hmC sites is significantly higher than that for the library-specific 5hmC sites (Figure S1E).
We further validated Aba-seq results using MspI and T4 β-glu-cosyltransferase (Kinney et al., 2011). We quantified 5hmC percentage at a number of CCGG sites with different read coverage in the 1CG1CH and 1CG1XX categories. A strong positive correlation between the read coverage and 5hmC percentage was observed (r = 0.73, Figure S1F). Background levels of 5hmC at random, unreported CCGG sites are below the detection limit (0.35% ± 0.56%). Levels of 5hmC for sites with read coverage equal to 1 and 2 are significantly higher than the background (mean = 2.2%, p value < 0.02, one-sided Student's t test), confirming the presence of 5hmC. The lowest detectable 5hmC percentage in CCGG sites was around 2%, giving an estimate on the sensitivity of Aba-seq. To validate 5hmC in the CH sites, we quantified AbaSI cleavage percentage at these sites by individual quantitative PCR (qPCR), as shown in Table S3. Similarly, all reported CH sites has significantly higher 5hmC readings than randomly chosen CH sites (p value < 1 ×10−9). Overall, our validation confirms Aba-seq resulted a high-quality hydroxymethylome of the E14 cells. In addition, regional 5hmC levels, as reflected by the cumulative read numbers in a genomic region, show strong correlation with the normalized 5hmC site density (Figure S1G). Therefore, we will only show results by using the 5hmC site density in subsequent analysis.
Comparison with the earlier hMeDIP data (Williams et al., 2011) suggests that 95% of the enriched peaks contain at least one Aba-seq 5hmC site. Importantly, the local 5hmC density of those sites outside the hMeDIP peaks is much lower than that of those inside (p value < 1 × 10−6, Figure S1H), which would suggest that the 5hmC antibody used in hMeDIP may favor the densely populated 5hmC regions. A recent study (TAB-seq) also reported base-resolution hydroxymethylome in mouse E14 cells with 2,057,636 5hmCs (Yu et al., 2012), about one sixth of our results in size. A comparison between Aba-seq and TAB-seq resulted in 1,246,351 common 5hmC sites (60.5% of TAB-seq data). Among our validated 5hmCG sites, 12 (~15%) were reported in the TAB-seq data set. A notable difference between the two methods is that TAB-seq may preferentially detect sites with high 5hmC occupancy (Yu et al., 2012). However, comparison in read numbers between Aba-seq-specific sites and the Aba/TAB-seq common sites yielded no significant difference. In addition, we compared our data and TAB-seq data with earlier base-resolution data from the single-molecule sequencing method (Song et al., 2011). Of the 744 hmCG sites reported by that method (Song et al., 2012), 522 (~70.2%) overlap with Aba-seq and 123 (~16.5%) overlap with TAB-seq. Further biological replicates and technical investigations are required to interpret the discrepancies between the different methods.
In ESCs, a significant amount (20%–30%) of 5mC exists in CH context, whereas this number reduces to negligible levels in somatic cells (Lister et al., 2009, 2011; Stadler et al., 2011). Based on our data, we estimate that 95.9% of all 5hmCs exists in CG context, whereas 4.1% exist in CH context (1.4% CHG and 2.7% CHH) (Figure 2A). These numbers differ from those reported by TAB-seq, where close to 99% of 5hmCG were observed (Yu et al., 2012). The average read number of the CH group (10.0) is significantly smaller than that of the CG group (12.4, p value < 1 × 10−6, two-sided Student's t test), suggesting lower 5hmC occupancy at the CH sites. Using 5hmCG sites with significant read coverage by a bionomial distribution (p = 0.5), we estimate that the percentage of the symmetrically hydroxymethylated sites within the 5hmCG sites exceeds 90% (Figure S2A), arguing against strand bias in hydroxylation of the fully methylated CG sites.
Examination of the 5hmC site density in chromosomes reveals comparable levels across the autosomes, with a slightly increase in chromosome 11 (Figure 2B). Sex chromosomes are depleted in 5hmC, with little 5hmCs in the Y chromosome (Figure 2B). Depletion of 5mC and 5hmC (Szulwach et al., 2011) in X chromosomes has been observed in neuronal tissues and stem cells. Together these results suggest a different epigenetic regulation mechanism between the autosomes and sex chromosomes. In addition, we observed the highest 5hmC site density in the mitochondria genome in both CG and CH contexts (Figure 2B). Considering that mitochondria genomes are present in multiple copies in the cell, it remains inconclusive whether they are enriched in 5hmC. Nevertheless, inside mitochondria DNA, normalized CH 5hmC site density is nearly as high as the normalized CG 5hmC site density, whereas it is less than 1% of the latter for all other chromosomes (Figure 2B). A set of key DNA epigenetic components, including DNMT1, TET enzymes, and their products 5mC and 5hmC, were reported in mitochondria (Shock et al., 2011). Our results further suggest that the prevalent pattern of hydroxymethylation, possibly methylation, in mitochondria genome might exist in an unbiased manner toward CG or CH. Overall, our results suggest a different epigenome in the mitochondria than that in the nucleus.
Most of the identified 5hmC sites reside in intergenic regions, followed by introns (Figure 2C). Compared with a base-resolution methylome map in the mouse ESCs (Stadler et al., 2011) (Figure S2B), 5hmCGs are slightly enriched in the intragenic regions and depleted in the intergenic regions. This is consistent with the previous findings using hMeDIP approaches (Ficz et al., 2011). A gene-centric view of 5hmC as well as 5mC distribution in both DNA strands is shown in Figure 2D. There is no noticeable difference for the 5hmCG distribution between the sense and the antisense strands (Figure 2D). We normalized the 5hmCG site density by the 5mCG density, which accounts for the local substrate concentration effect presented to the TET enzymes. The normalized 5hmCG site density is reduced at the transcription start sites (TSSs) and 5′ UTR regions (Figure 2D), and a similar trend is observed for the 5hmCH site (data not shown). Interestingly, although the absolute 5hmCG and the 5mCG densities are higher in exons than in introns, the normalized 5hmCG site density does not show difference between exons and introns (Figure 2D). Overall, our results suggest that the 5hmC profile closely follows the 5mC distribution in the genic regions (Lister et al., 2009).
Similar to 5mC, 5hmC is depleted in the CpG islands (CGIs) at the genome scale (Figure 3A). Levels of 5hmC in CGIs, as estimated by the cumulative reads, are inversely correlated with the CpG density (Figure S2C), consistent with an earlier report (Booth et al., 2012). We manually separated all CGIs into two groups based on the overall ranked 5hmC density: group I (top 5%) and group II (lower 95%) (Figure 3A, middle panel). In group I, even with the increased 5hmC density inside CGIs, there is no significant enrichment compared with the flanking regions (data not shown). Most of the group I CGIs appear in exonic regions, whereas most of the group II CGIs appear in the TSS/promoter regions (Figure 3A). The higher 5hmC density in exonic CGIs may be due to their higher 5mC levels compared with the TSS/promoter CGIs (Lister et al., 2009; Deaton and Bird, 2011).
An observed enrichment of 5hmC in repetitive DNA, such as LINE elements, has been previously reported (Ficz et al., 2011). However, our results show that the genome-averaged 5hmC density inside LINE is at the same level as their flanking regions (Figure 3B). Detailed analysis separated all LINE elements into three categories: (1) those that do not contain CpG sites inside the elements (~25%); (2) those that contain CpG sites but no 5hmCG sites (~42%); and (3) those that contain both CpG sites and 5hmCG sites (32%). Although category II can be regarded as depleted of 5hmCG, there is moderate but significant 5hmCG enrichment in category III (Figure 3B, middle and lower panel). Similar patterns were observed for the SINE and LTR repetitive elements (Figure S3). Overall, our results argue that there is only a small fraction of repetitive elements showing 5hmC enrichment in ESCs.
5mC is generally depleted at the cis-elements to facilitate transcription factor (TF) binding (Lister et al., 2009). By correlating with a set of chromatin immunoprecipitation sequencing (ChIP-seq) data, Figure 4A illustrates the representative 5hmC distributions around various protein-DNA interaction sites (Chen et al., 2008).
The first intriguing observation is a symmetrical, regularly spaced oscillating distribution of 5hmC around the CTCF binding sites in both CG and CH context (Figure 4A). The center of the CTCF binding sites are slightly CG-rich (Figure 4A, CpG line) and depleted in 5mCG (Figure 4A, 5mCG/CG line), consistent with the methylation sensitivity for the CTCF binding (Kurukuti et al., 2006). Accordingly, 5hmCG and 5hmCH are depleted at the center of the CTCF binding sites (Figure 4A). The distance between the neighboring 5hmC peaks on either side of the binding site is about 150 nucleotides (Figure 4A), reminiscent of the unit length of the nucleosome-wound DNA. It was suggested that CTCF binds to the linker DNA between the nucleosomes and the binding event leads to highly organized local nucleosome array on both sides (Fu et al., 2008; Cuddapah et al., 2009). The observed regularity of the 5hmC distribution is in line with such local nucleosomal structure. This further suggests that the local nucleosome environment may play an important role in defining the 5hmC profile, possibly by affecting the 5mC accessibility to the TET enzymes.
A second interesting observation is for the Nanog binding sites, which show noticeable accumulation of 5hmCH at the center, but not 5hmCG (Figure 4A). Similar patterns are also observed for Sox2, Oct4, STAT3, Esrrb, and Tcfcp2I1 (Figures 4A and S4). Nanog/Oct4/Sox2 are known to co-occupy a same set of loci in ESCs and their predicted binding motifs do not contain CG dinucleotides (Chen et al., 2008). To investigate whether non-CG 5-hydroxymethylation affects binding, we then compared the binding affinity of Sox2 to the unmodified, methylated, and hydroxymethylated sites (Figure S5). Our results suggest that cytosine modifications within the binding site do not affect Sox2 binding. In addition, structural modeling reveals that Sox2 binds DNA in the minor groove, whereas the five-position modification of cytosine is located in the major groove and thus not recognized (Figure S5). Further investigation is needed to explain the non-CG accumulation of 5hmC around the binding sites of these transcription factors.
For the other factors we studied, including c-myc, nmyc, Klf4, E2f1, Zfx, and 5hmC, distribution was either slightly depleted or relatively constant around the binding sites (Figures 4A and S4). Binding sites of the histone acetyltransferase p300 showed unique fluctuating 5hmC distributions (Figure 4A).
We next analyzed the 5hmC distribution around the ChIP-seq peaks of various chromatin modification marks (Mikkelsen et al., 2007), as shown in Figure 4B. Both 5mC and 5hmC are depleted in regions marked with H3K4me3, an activation mark. Furthermore, 5hmC is depleted in the RNA polII (Mikkelsen et al., 2007) occupied regions (Figure S4), suggesting 5hmC is generally excluded from active transcriptions. For regions marked with H3K27me3, a repression mark, 5hmC does not show obvious depletion or enrichment. Previous reports have documented 5hmC enrichment in enhancer regions (Stroud et al., 2011; Szulwach et al., 2011). We investigated a few histone marks that are associated with enhancers. For the H3K27Ac marked regions, which may act as active enhancers (Creyghton et al., 2010), 5hmC is slightly depleted as well as 5mC. In contrast, for the H3K4me1 marked regions, which may indicate poised enhancers (Creyghton et al., 2010), both 5mC and 5hmC are enriched toward the center of the regions. Lastly, both 5mC and 5hmC are enriched in the H4K20me3 marked regions, which are associated with specific classes of repetitive elements (Martens et al., 2005).
The epigenetic processes of 5hmC regulation remain elusive and controversial after its rediscovery. Base-resolution genome-wide hydroxymethylomes as well as methylomes provide the first steps toward probing their biological relevance. Compared with other bisulfite-conversion based methods, Aba-seq has several unique advantages: (1) simple library construction even with low amount of input DNA (100 ng in our experiment); (2) 5hmC sites with low occupancy can be reliably detected; and (3) data processing is more straightforward. Its potential limitations, on the other hand, largely stem from the enzymatic properties of AbaSI (Wang et al., 2011). For example, target sites are potentially cleaved with varying efficiency, with 5gmC at both sides of the cleavage sites showing the highest efficiency (Wang et al., 2011). However, this appears not a big concern as such sites are only slightly overrepresented in our current data set (Figure S1I). For a small portion of data (~13% in our data set), there is ambiguity in assigning 5hmC to the exact base. Nevertheless, approximately 12.2M putative 5hmC sites are detected in the mouse ESC genome and bioinformatic analysis has provided sufficiently fine details about the E14 hydroxymethylome.
Based on our results, we propose that factors affecting the local 5mC accessibility to the TET enzymes play important roles in the 5hmC deposition. These factors can include chromatin compaction, nucleosome positioning, or TF binding. The most striking example is the regularly oscillating 5hmC profile around the CTCF-binding sites, suggesting 5hmC "writers" may be sensitive to the nucleosomal environment. Similarly, the elevated 5hmC levels in the exonic regions are possibly due to a higher concentration of more accessible 5mC sites. The 5hmC level after normalization is actually comparable for the inter- and intragenic regions, arguing against specific enrichment of 5hmC in either region. Depletion of 5hmCs is often observed when there is depletion of 5mC, as seen in the TSS regions, binding sites of RNA polII, and some TFs. For 5hmCs in other regions—in particular, poised enhancers marked by H3K4me1—the presence of 5hmC may indicate a transition or a poised state between methylation and demethylation. Finally, the unique mitochondrial hydroxymethylome and the locally increased CH hydroxymethylation at TF-binding sites such as NANOG and SOX2 await further biological investigation.
The undifferentiated E14 cells was cultured as previously described (Kinney et al., 2011). E14 cells were harvested and DNA extracted with a QIAGEN Pure-gene Core Kit (catalog no. 1042601) per manufacturer's specifications. E14 genomic DNA was then treated with QIAGEN RNase A (catalog no. 19101).
Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4. Glucosylated genomic DNA was subsequently purified by phenol-chloroform extraction and ethanol precipitation. AbaSI digestion was done by incubating the glucosylated genomic DNA with four units of AbaSI in NEB buffer #4 at room temperature (25°C) for 1 hr in a total reaction volume of 20 μl. The AbaSI digested DNA was then purified by phenol-chloroform extraction and ethanol precipitation. Biotinylated P1 adapters (Table S1) were ligated onto the AbaSI digested DNA by incubating the digested DNA with 300 nM of P1 and 2 μl of T4 DNA quick ligase (NEB: M2200) in the 1 × quick ligation buffer in a total volume of 42 μl at room for 30 min. Ligated DNA was then purified by running samples on a 6% Tris-borate-EDTA (TBE) gel followed by gel excision of the DNA. Gel extraction was done by crushing and soaking the isolated gel fragment (0.3M NaOAC [pH 7.4], 2 mM EDTA) overnight at 37°C followed by ethanol precipitation of the DNA. Covaris s-series sonicator was used to shear the P1-ligated genomic DNA to a range around 300 bp with the following settings: duty, 10; intensity, 4; cycles per burst, 100; six cycles at 60 s. P1-ligated DNA was then captured by mixing 50 μl of fragmented DNA with 12.5 μl of Dynabeads MyOne Streptavidin C1 beads (Life Technologies, catalog no. 650–01) according to the manufacturer's specifications. End repair was done by using the NEBNext End Repair Module (NEB: E6050S) on the DNA-bound beads for 30 min at 20°C. The DNA-bound beads were then washed two times with a buffer containing 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, and 1 M NaCl; followed by wash with H2O. A-tailing of the end-repaired DNA on beads was carried out by using the NEBNext dA-Tailing Module (NEB: E6053S) for 30 min at 37°C. The DNA-bound beads were then washed as the previous step. P2 adapters (Table S1) were ligated by incubating the DNA-bound beads with 250 nM of P2 and 2 μl of T4 DNA quick ligase in the 1× quick ligation buffer at room temperature for 30 min. The DNA-bound beads were then washed as the previous steps. Amplification of the DNA library was carried out by adding the entirety of the DNA-bound beads to a reaction mixture containing 0.2 mM of each dNTP, 1× Phusion HF Buffer, 300 nM forward primer (PCR_I), 300 nM reverse primers (PCR_IIpe) (Table S1), and ten units of Phusion DNA polymerase (NEB: M0531S). The following thermocycler conditions were utilized for amplification: 30 s 98°C, 16 cycles (10 s 98°C, 15 s 70°C, 15 s 72°C), 10 min 72°C, and hold at 4°C. The beads were then removed from the PCR and the library was extracted by gel purification as described above.
E14 genomic DNA used in this assay was same batch to that of the Aba-seq library construction. Validation of genomic 5hmC at CCGG sites were carried out by using the EpiMark 5hmC and 5mC Analysis Kit according to manufacturer's specifications (NEB: E3317S). Real-time PCR of digested DNA was done with iQ SYBR Green Supermix (BIO-RAD: 170–8882) per manufacturer's specifications.
Validation of genomic 5hmC at CH sites were done on AbaSI-digested glucosylated E14 genomic DNA followed by real-time PCR. Real-time PCR of digested DNA was done with iQ SYBR Green Supermix (BIO-RAD: 170–8882).
All real-time PCR primers and the method used to calculate 5hmC percentage are listed in Table S2 for CCGG sites and Table S3 for CH sites.
All sequencing runs (two lanes) were done on the Illumina HiSeq platform with read length of 50 bp. All the raw reads were mapped to the mouse reference (UCSC mm9) using the Bowtie (Langmead et al., 2009) aligner with parameters (-v 2 -m 1). We then interrogated neighboring sequences of the aligned AbaSI cleavage sites in search of putative 5hmC sites at expected distance (−CN11–13 or +N9–11G). Copy number is defined as the total number of reads that resulted from AbaSI digestion of a particular 5hmC site. Sequence logos were constructed using weblogo (Crooks et al., 2004). All scripts were written in PERL and are available upon request.
CpG methylome of mouse ESC by Bisulfite sequencing (BisSeq) was acquired from NCBI Gene Expression Omnibus (GEO) database GSE30202 (Stadler et al., 2011) and was used as a reference. 99% (11,066,961) of Aba-seq 5hmCG sites overlap with the 5mCG sites in this BisSeq data set. The 11,066,961 overlapped 5hmCG sites were used for the subsequent genomic analyses of 5hmCG in this study.
All genomic regions and their genomic coordinates were defined based on the mm9 genome annotation tracks downloaded from UCSC Genome Browser (Fujita et al., 2011) in February 2012. Different genomic elements were identified from the RefSeq genes track. Promoter is defined as 5 kb upstream of nonredundant TSS. We define intergenic regions as 5 kb downstream of transcription termination sites (TTS) that do not overlap with any known genic regions.
To generate genome-wide metagene plots we divided 2 kb upstream region of TSS, 5′ UTR, exon, intron, 3′ UTR, and 2 kb region downstream of each nonredundant RefSeq gene into equal-sized bins respectively. For CGIs and repetitive elements, we binned the same-length upstream and downstream flanking regions into same number of intervals. CpG, 5mCG, 5hmCG, and 5hmCH sites were mapped to individual regions using BEDTOOL (Quinlan and Hall, 2010). Site density was calculated as number of sites divided by region length. 5hmCG density was also corrected by background CpG and 5mCG. Average densities were then computed for each bin position and were used for metaplots or global trend plots.
Genomiccoordinates of the CGIs wereacquired from the cpgIslandExt table in UCSC database. Depending on their locations, we classified CGIs into promoter CGI, TSS CGI, exon CGI, intron CGI, 3′ UTR CGI, and TTS CGI (a TSS or TTS region is defined as −500 bp to +500 bp of a TTS or a TTS). Each type of CGIs has at least 50% overlap with a region of that particular genomic feature.
Repetitive elements (LINE, SINE, and LTR) were retrieved from the Repeat-Masker track in UCSC database.
We compared our Aba-seq data set to three different types of 5hmC mapping data sets: hydroxymethylated DNA immunoprecipitation sequencing (Williams et al., 2011); selective chemical labeling of 5hmC with single-molecule real-time DNA sequencing (SMRT) (Song et al., 2012) and TAB-seq (Yu et al., 2012).
All the above data sets were converted to BED format. Nonredundant 26-mer fragments flanking the AbaSI cleavage sites were used to compare with the 5hmC-enriched regions derived from hMeDIP data. Comparison between AbaSI-seq, TAB-seq, and SMRT were carried out at base resolution by BEDTOOL.
To create 5hmC profile at ChIP-seq peaks, we downloaded data sets from NCBI.
A region of −1 to +1 kbp around the peak summits or the binding centers was selected and binned into 50 bp sliding windows at 25 bp steps. 5mCG, 5hmCG, 5hmCH, and CpG sites were aligned to individual bins and site density profiles were normalized to the site density per 1 bp per bin. Global trend plots were generated by calculating the average site density at each bin position.
We thank Dr. Shannon Kinney for providing E14 genomic DNA, Drs. Rich Rob-erts and William Jack for helpful comments, and Drs. Peng Jin and Chuan He for providing us TAB-seq dataset of mouse E14 cells. We thank Cynthia Hen-drickson and HudsonAlpha Institute for Biotechnology for sequencing. This work is supported by NIH SBIR grants (GM096723 to Z.Z. and GM095209 to Y.Z.) and New England Biolabs. Subjects of this paper may be potential future products of New England Biolabs.
All data have been submitted to the GEO database under the accession number GSE42898.
Supplemental Information includes five figures and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2013.01.001.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Total number of Fido stores in North Vancouver: 1
The biggest mall in North Vancouver with Fido store: Capilano Mall - North Vancouver
Fido stores in North Vancouver - get information about the brand Fido and all Fido locations in North Vancouver - British Columbia, Canada.
Select Fido store in North Vancouver from the list below. All Fido stores are also displayed on the map.
300 Borough Drive, Scarborough, Ontario, M1P 4P5, Canada198 stores
25 The West Mall, Toronto, ON, M9C 1B8213 stores
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CORBERO EN400IN/2 Owner's Manual
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After fresh ideas for meals that are healthy but still pack a flavour punch? We've got salads and vegetable-packed bowls to soups and light desserts.
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These three restaurants - Fleet, Brae and Igni - might not be in capital cities, but their food is all the more satisfying for the time it takes to travel there.
The life of a farmer revolves around the seasons. Come winter, a certain thriftiness is needed in the kitchen to make the most of meagre produce, writes Paulette Whitney.
Italy's claim to being the greatest of the world's cuisines has one key weakness: breakfast. But, argues John Irving, there's more to the story than first meets the eye.
The hottest spots to eat, drink, play and stay on your next trip to LA, rounded up into one perfect day.
Your guide to a perfect stay in Canberra, from where to sleep to the exhibitions you need to check out.
Some of Australia's best dining destinations take the hassle out of a weekend stay by offering their own on-site digs where you can hit the hay in style after your meal.
The maitre d' is your first introduction to a restaurant - they do as much to create a sense of ambience as lighting, tableware and music. And these three professionals are top of the class.
Three sommeliers, three different personalities, all first-rate guides to the lists at their establishments. We present our 2018 finalists: Caitlyn Rees, Gaving Cremming and Patrick White.
Kicking off in February 2018, six exclusive tours will take Gourmet Traveller readers far and wide, delivering exceptional service, fine dining and, of course, a first-class travel experience.
The restaurant and hotel scene on Australia's favourite holiday island has never been more exciting and Australian chefs, owners and restaurateurs are leading the charge, writes Samantha Coomber.
It's the most popular coffee in Australia, but what is a flat white exactly? Samantha Teague investigates.
The chef at Bistrode CBD and The Fish Shop passed away today, 17 July 2017.
Ambling through a forgotten corner of the country offers a charming change of pace from Lisbon and the Algarve.
Just what you need on a cold winter's night; a bowl of luscious pudding. Make sure to leave room for seconds.
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Teage Ezard's Black restaurant has barely opened and yet the bar
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apéritif. The Leisure Suit's name, says bartender Michael Chiem,
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The Star, 80 Pyrmont St, Pyrmont, NSW, 1800 700
Muddle a thick slice of cucumber in the bottom of a shaker or tin, add ice, 50ml Plymouth gin, 15ml Védrenne elderflower cordial, 30ml cloudy apple juice and a very small dash of rosewater and shake hard. Pour through a fine strainer into a chilled cocktail glass, garnish with dried rosebuds and serve frosty.
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Lyrics Depot is your source of lyrics to More Than A Bedroom Thing by Bill Anderson. Please check back for more Bill Anderson lyrics.
More Than A Bedroom Thing Lyrics
Artist: Bill Anderson
Album: Nashville Mirrors
There's nothin' I like any better than turnin' the lights down low
Turnin' the covers back and lettin' all of our feelin' show
My favorite room is the bedroom my favorite time is night
My favorite place is in your arms nothin' lovin' and holdin' you tight
But our love is more than a bedroom thing
It strats there but it don't stop there it's an old and compassing thing
You're a fantastic woman in the kitchen a dynamite lady in the den
You're a hell of a lover in the bedroom but our love is more than a bedroom thing
So much more more than a bedroom thing
[ guitar ]
Sometimes I get high just a sittin' and talkin' to you
Laughin' at the things we done and plannin' the thing we goin' to do
Your fingers touchin' my body your eyes touchin' my soul
Your kisses leading me back to the room where the waters of passion flow
But our love is more...
Bill Anderson Lyrics
Bill Anderson Nashville Mirrors Lyrics
More Bill Anderson Music Lyrics:
Bill Anderson - First Mrs Jones Lyrics
Bill Anderson - Footprints In The Sand Lyrics
Bill Anderson - I Don't Have Anyplace To Go Lyrics
Bill Anderson - In The Misty Moonlight Lyrics
Bill Anderson - Knowing You're Mine Lyrics
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Bill Anderson - Take My Hand Precious Lord Lyrics
Bill Anderson - Who Is The Biggest Fool Lyrics


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Formule 1 Hotels in Delhi
(2 Hotels in Delhi)
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Choose from below List of Available Formule 1 Hotels in Delhi for Online Hotel Booking
Room: size of room was not big but decent only. WiFi connectivity was good Location: very close to market Hospitality: staff were cooperative Food : taste nice but limited option View: nice surrounding
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Couple Traveller
Rooms are good but quite smaller than they appear in the pictures as displayed. They were clean and food was good but quite expensive. Room service was good as they were quite quick. Only problem is the rooms were very small.
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A longstanding customer at a major US bank recently remarked to me, âYou can take AutoMate out of the IT department, and should.â While the usage of AutoMate and BPA Server outside of IT organizations is not news (about 30% of our deployments are outside of IT), the notion that âwe shouldâ poses some interesting opportunities, but also dilemmas.
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This is, hands down, the BEST banana cake I've ever had. It's soft, fluffy, moist and rich all at the same time! Once cooled this cake is topped with a totally irresistible lemon cream cheese frosting for a perfect dessert your family will love.
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These Soft and Chewy Sugar Cookie Bars are the perfect kid-friendly dessert that the whole family will love! Recipe from thebusybaker.ca!


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Home Alone may not be your typical home renovation flick, but we can't deny that Kevin (Macauley Culkin) certainly "upgrades" the McCallister home to suit his needs. After being accidentally left behind during a family vacation to France for Christmas, Kevin gets to live his fantasy of having the whole house to himself! The glee is short-lived, however, as Kevin soon encounters a pair of burglars who are determined to ransack his home. How does he respond? With a little mischievous home improvement.
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Take Action Tour at Revolution, April 30, Benefits Teen Sex Ed
Nothing like a little good-doing to offset the sex-and-drugs image of touring rock and roll bands! For almost a full decade now (!) Hopeless Records has been fighting this good fight, sending out its Take Action Tour around the country, alongside a compilation on its own nonprofit label, Sub City, to raise additional funds. The tour and comp rotate a cause each year, but it's always something close to benefiting the kids who turn out to the shows.
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Jordan Taylor Bunch
Jordan is a disciple-maker and simple church planter, although his favorite title is "Sara's husband." He also works as a wedding photographer, film maker, writer and Christian small group trainer. Jordan earned his B.A. and M.A. in Domestic Missions at Abilene Christian University. He has a deep passion for seeing the Church become everything She was meant to be, and thus has made it his mission in life to join Jesus as He goes about restoring His Bride.
Sara Elise Bunch
Sara is a gifted host and includer. She makes everyone feels welcome and loved where ever she goes. She has a deep passion for those on the margins of society. She graduated with her B.S. and Masters of Communication Sciences & Disorders from Abilene Christian University. She works full time in early childhood intervention as a Speech Language Pathologist with children from birth to age 3 and their families.
"But you will receive power when the Holy Spirit comes on you; and you will be my witnesses in Jerusalem, and in Judea and Samaria, and to the ends of the earth." ~King Jesus (Acts 1:8)


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Only the greatest person to ever play Final Fantasy XI, he was so cool, if i had a dollar for every person thats not as cool as him i would be rich.
by Matt December 09, 2004
The Urban Dictionary Mug
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Rainbow Reading Level 5 - Move Your Body: My Blue Takkies Box E
Written by Cheryl Minkley
Illustrated by
Rainbow Reading Level 5 - Move Your Body: My Blue Takkies Box E by Cheryl MinkleyThis light-hearted story features a pair of blue takkies that transport the main character into some very unusual situations where she meets some odd fantasy characters.
About the Author
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Book Info
24 pages
Cheryl Minkley
More books by Cheryl Minkley
Cambridge University Press
Publication date
13th March 2009
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With iPad Sales Falling, Apple Pushes Into Businesses
Tech giant is working with more than 40 companies to make the iPad a more appealing work tool
Apple Inc., grappling with swooning sales of the once-blockbuster iPad and sensing an opening in the changing ways of work, is making its broadest assault to date on business computing.
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Dubba Jonny
Who Shot JR?
Mrs Mangle Presents....
Arrrgggghhh we've created a monster....
It's amazing how things can spiral and take on a life of their own. What started as the simple idea of a party for Mrs Mangle's birthday, has now transformed into an evening of unadulterated bass.
In hopefully (fingers crossed) the first of many nights, Mrs Mangle invites you to come share the celebrations while having your ears assaulted by some of the dirtiest tunes known to man.
And who better to lead such assault, than none other than Stench man, created in 1985 by rogue scientists trying to splice the genes of a Friesian cow and a severely impaired young gypsy. Stenchman has been destroying dance floors around the world with his dirty take on the Dubstep genre.
Not only that, weve managed to drag Dubba Jonny (plus Drumkit) up for the night! A Bournemouth based 2 man operation, consisting of producer Brian (also known as 'Kuvah' of Duke & Kuvah) and drummer Nick. Their shows combine Dj'ing, synth and sampling with live drumming for an extra explosive set.
Warming up to get you in the mood is Mrs Mangle's Resident Support "Who Shot JR" providing some filthy electro bass shizzle to get the party started!
Line Up
Stench man
Dubba Jonny
Who Shot JR?
53 Degree's (upstairs)
Brook St, Preston
Friday 7th May 2010
10pm till 3am
Advance tickets £6 / More on the Door
Available from UCLAN SU / Action Records
Dresscode : No Tracksuits. Trainers ok
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