{'fraction_non_alphanumeric': 0.027932270423927822, 'fraction_numerical': 0.025336793968607094, 'mean_word_length': 4.851048445408532, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Large Differences in Small RNA Composition Between Human Biofluids

SUMMARY Extracellular microRNAs (miRNAs) and other small RNAs are implicated in cellular communication and may be useful as disease biomarkers. We systematically compared small RNAs in 12 human biofluid types using RNA sequencing (RNA-seq). miRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads in all biofluids, but the ratio of miRNA to tDR reads varied from 72 in plasma to 0.004 in bile. miRNA levels were highly correlated across all biofluids, but levels of some miRNAs differed markedly between biofluids. tDR populations differed extensively between biofluids. Y RNA fragments were seen in all biofluids and accounted for >10% of reads in blood plasma, serum, and cerebrospinal fluid (CSF). Reads mapping exclusively to Piwi-interacting RNAs (piRNAs) were very rare, except in seminal plasma. These results demonstrate extensive differences in small RNAs between human biofluids and provide a useful resource for investigating extracellular RNA biology and developing biomarkers.

central nervous system. Lumbar punctures were performed for research purposes only. CSF was collected in polypropylene tubes and placed immediately on wet ice for transport to the UCSF Core Virology Laboratory where it was processed within 1 h. After centrifugation at 600 x g for 10 min to remove cells, supernatants were aliquoted and snap frozen for storage at -80°C.

Ovarian Follicle Fluid
Healthy egg donors underwent ovarian follicular stimulation with gonadotropins per UCSF clinic protocols. Ultrasounds were performed to assess and confirm uterine lining and ovarian follicle maturation. Approximately 36 h after human chorionic gonadotropin injection when a sufficient number of mature-sized follicles had developed, the follicles (≥16 mm diameter) were aspirated for egg retrieval using negative pressure into 10 mL tubes prior to transvaginal ultrasoundguided oocyte retrieval. Two independent samples of 3.5-5.0 mL of follicular fluid were collected from each study participant, one from each ovary. Visual inspection and Combur-9-test urine sticks (sensitivity 5 erythrocytes/µL) were used to exclude samples with blood contamination.
Each sample was centrifuged at 400 x g for 10 min at room temperature. Supernatants were aliquoted and immediately frozen at -80 °C.

Adult Blood Plasma, Serum, and Urine
Blood and urine were collected from healthy adults ages 18-70 with no evidence of coronary heart disease, peripheral vascular disease, congestive heart failure, cerebrovascular disease, cancer, chronic lung disease, endocrinopathy, or renal disease by subject report or by history and physical examination. Subjects fasted for a minimum of 8 h prior to blood and urine collection. Blood was collected using a 21-gauge needle and the first 5 ml was discarded.
Plasma was prepared from EDTA-containing vacutainer tubes via centrifugation for 10 min at 500 x g at 23 °C, followed by transfer of the top layer to fresh tubes (avoiding buffy coat), followed by centrifugation for 10 min at 2000 x g at room temperature and transfer of the top 80% of the plasma to fresh tubes for storage at -80°C. Serum was collected from red top vacutainer tubes that were kept at room temperature for 30-45 min to allow for coagulation, followed by centrifugation for 10 min at 2000 x g at room temperature. Urine was collected by clean catch in a sterile collection container, followed by centrifugation for 10 min at 2000 x g at room temperature. The top 80% was transferred to fresh tubes followed by storage at -80°C.

Saliva
SMSL and parotid saliva were collected as described (Albertolle et al., 2015). Briefly, subjects thoroughly rinsed their mouths with water prior to sample collection. Salivary flow was stimulated by the application of citric acid to the tongue. Parotid secretions were obtained by using a Lashley cup. SMSL saliva was obtained by using a Block and Brotman collector that fit around the gland openings. The secretions were collected on ice. Immediately thereafter, a protease inhibitor cocktail (Pierce) was added and samples were briefly vortexed, divided into 1 mL aliquots, and frozen at −80 °C.

Seminal Plasma
Semen samples were collected through masturbation into a sterile cup and centrifuged to remove sperm. Semen analysis was performed according to WHO 2010 guidelines in the UCSF Center for Reproductive Health Andrology Laboratory. All study participants had normal semen parameters. Samples were centrifuged for 5 min at 428 x g and supernatants were stored at -80°C.

RNA Isolation, Library Preparation, and Sequencing
The small RNA-sequencing method used to generate libraries relies on a version of the TruSeq Small RNA Library Preparation Kit modified by using randomized adapters, adding PEG to the adapter-RNA solution, and including steps to enzymatically remove excess adapter after 3' ligation. Multiple libraries with unique indexes were pooled, purified using the Qiagen MinElute PCR Purification Kit per the manufacturer's recommendations. Libraries were size-selected using the PippinPrep (Sage Science) with a 3% agarose gel. In pilot experiments, we adjusted the size selection parameters to maintain a low proportion of adapter dimers (132 bp) and maximize the proportion of library with inserts of ~22-30 bp. We selected the PippinPrep broad range option to deplete sequences <137 bp and >166 bp but maintain a range of insert sizes within this range. Size-selected DNA was sequenced on an Illumina HiSeq 4000 (single end 50 base mode).

qPCR validation
Samples collected for qPCR experiments were obtained from healthy subjects enrolled in a study conducted at the University of California, San Francisco. Samples were collected from 11 males and 8 females. Of these 19 subjects, 1 is Native America, 3 are Hispanic, 4 are Asian, and 11 are Caucasian. The median age was 32 years (interquartile range, 27.5 -42.5). For BAL fluid collection, segmental lavage with two 50 mL aliquots of warmed sterile normal saline in each of two segments for a total instilled volume of 200 mL with typical return ranging from 80-120 mL. Lavage fluid was filtered through 2-ply gauze. BAL fluid was collected into 15 mL polypropylene conical tubes and centrifuged at 300 x g for 5 minutes at 4°C. 1 mL of BAL supernatant was aliquoted into 1.5 mL Eppendorf O-ring tubes and stored at -80°C. For collection of adult blood plasma, blood was collected in K2 EDTA purple top tubes using a 21gauge needle. Tubes were centrifuged at 1000 x g for 10 minutes, aliquoted into 250 µL volumes, and stored at -80°C. 400 µL of BAL fluid was extracted with the Qiagen miRNEasy Mini kit as per the manufacturer's protocol and eluted in 50 µL water. 200 µL of adult blood plasma was extracted with the mirVana PARIS kit as per the manufacturer's protocol and eluted into 100 µL water. 2 µL of input RNA was used for reverse transcription.
We performed quantitative PCR (qPCR) for selected miRNAs using methods described previously (Moltzahn et al., 2011 andSeumois et al., 2012). In brief, we applied a stem-loopbased multiplex quantitative reverse transcription PCR method and pre-amplifications (14 cycles), followed by purification of multiplex PCR products and subsequent uniplex analysis on a microfluidics chip (Fluidigm, South San Francisco, CA). An amplification curve quality score of ≥ 0.5 was required; otherwise, the reaction was considered to have failed amplification.
Technical duplicates were run and Ct's averaged as the initial step. Samples then underwent global mean normalization (Mestdagn et al., 2009) using all miRNAs that yielded a Ct in any given sample. As all of the above steps were done on Ct data, the normalized expression values were considered to be on a log base 2 scale. The ratios plotted in Figure S2 were calculated by subtracting BAL fluid normalized values from adult blood plasma values within each platform. To generate p-values, we performed Student's t-test for each miRNA. Pvalues were adjusted by FDR to correct for multiple testing.