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FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C

Summary The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).


SUMMARY
The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).

BEFORE YOU BEGIN
The Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) method is based on the construction of a next-generation sequencing library using the transposase Tn5 (Buenrostro et al., 2013;Chen et al., 2016). The transposase enables the generation of the ATAC-seq library by simultaneous fragmentation of chromatin and integrating specific adapters into open chromatin regions. Genomic regions with accessible or open chromatin can be sequenced by next-generation sequencing and analyzed using bioinformatics tools. Most of the current epigenetic approaches allow profiling of chromatin landscapes from naive nuclei (Buenrostro et al., 2013, Buenrostro et al., 2015. Fixation and mechanical homogenization of the tissue tend to reduce the quality of the data. Here, we present an ATAC-seq method capable of generating high-quality chromatin organization and accessibility data from formaldehyde-fixed nuclei. The protocol includes several steps, such as extraction of the mouse cortical nuclei in low sucrose buffer and centrifugation in gradient iodixanol solutions, and immunolabeling and sorting of NeuN-positive nuclei for further analysis of chromatin structure (Hempel et al., 2007;Marion-Poll et al., 2014;Javier Rubio et al., 2014;Policicchio et al., 2020). Hi-C (high-throughput chromosome conformation capture) is a method to study 3-D genome structure and gene regulation, which facilitates the understanding of the correlation between chromatin organization and gene expression (Rao et al., 2014). Fixation is a critical step to preserve nuclear and cellular architecture. In Hi-C, DNA-protein complexes are fixed with formaldehyde, causing interacting loci to be bound to one another with covalent DNA-protein cross-links. Our modifications allow us to use a reduced amount of starting material, compared to the original paper (Belaghzal et al., 2017). Moreover, we optimized the current protocol for ATAC-seq and Hi-C for a single mouse cortex.
We recommend that you conduct nuclei isolation, sorting and several first steps of ATAC-seq and Hi-C in one day in order not to freeze nuclei (steps 28-31 for ATAC-seq and 52-69 for Hi-C). Then, continue experiments in parallel. The minimum is 4 days to finish library preparation. Of course, you can distribute all steps as it is convenient for you.

STEP-BY-STEP METHOD DETAILS
Timing: 2 h 30 min for 1 mouse Isolation of nuclei from the mouse brain cortex (10 months old, C57/BL/6J) ( Figure 1) Note: This protocol describes the procedure for 1 mouse. Increase time by the number of mice to be processed.
1. Mouse is sacrificed with isoflurane and perfused with cold PBS. Cortex is isolated on the ice Note: We do not recommend keeping the isolated cortex more than 10-15 min on ice.
2. Homogenize cortical tissue in 500 ul of ice-cold low sucrose buffer using Dounce homogenizer 10-15 times on ice. 4. Add glycine to homogenate to get 0.2M glycine solution, mix gently and incubate for 10 min at 22 C temperature to quench the crosslinking reaction.
Note: In the case of multiple samples, it is important to keep them on ice after this step until the next one.
5. Centrifuge the samples for 5 min at 2000 g at 4 C, discard the supernatant. 6. Resuspend the pellet in 3 mL of low sucrose buffer with PIC. 7. Homogenate each sample with the mechanical homogenizer (rotor/stator) at 15 rpm 4 times.
Each time is about 6 s. 8. Centrifuge the samples for 5 min at 2000 g at 4 C, discard the supernatant. 10. Resuspend pellet from step 8 in 2 mL of 22% iodixanol solution and prepare a gradient. Place in a 15 mL tube 1 mL of 43% iodixanol solution, then carefully add a layer of 1 mL of 22% iodixanol solution, on top add 2 mL of tissue homogenate in 22% iodixanol solution. 11. Centrifuge at 2600 g for 40 min at 4 C on the bucket centrifuge.

CRITICAL: Usage of a bucket centrifuge is necessary
Note: You can increase the time of centrifugation if needed.
12. Collect the nuclei interphase between 43% iodixanol solution and other layers. 13. Dilute the nuclei interphase fraction with resuspension solution with 1% BSA (1:2) 14. Centrifuge at 3200 g for 10 min at 4 C, discard the supernatant. 15. Wash the pellet once with 1 mL of resuspension solution with 1% BSA 16. Centrifuge at 3200 g for 5 min at 4 C.

FACS staining for sorting
Timing: 1 h 15 min for 1 mouse 17. Discard the liquid and resuspend the pellet in 200 mL PBST with 5% BSA and 3% goat serum.  100,000 nuclei were collected from the pellet (step 27) for the transposition reaction.
Note: The number of nuclei might be reduced to 50,000.
CRITICAL: Since we perform ATAC-seq on fixed nuclei it's crucial to do this step. It helps to achieve a good quality of ATAC-seq library.
31. Incubate the mixture at 65 C without shaking for 4-5 h. Repeat this step again. 46. Separate the beads on the magnet, discard the solution. 47. Leave the beads on the magnet for 5-10 min to air-dry the beads from the ethanol. 48. Remove the tube from the magnet.  49. Add 10 mL of 10 mM Tris-HCl pH 8.0 to the beads to elute the DNA. Mix gently by pipetting. The volume of 10 mM Tris-HCl pH 8.0 depends on the volume of the initial sample and your needs, but the minimum is 10 mL. 50. Incubate for 10 min at 22 C. 51. Place the tube on the magnet for 2-3 min. Transfer the solution to a fresh tube.

Timing:~4 days
Collected nuclei were used for downstream applications such as Hi-C (Figure 4). Hi-C was performed as previously described (Belaghzal et al., 2017) with minor modifications. 52. About 1,2-1,4 million of fixed neuron nuclei were collected from step 3. 53. Add 11 mL of 103 DpnII buffer (up to 1.13), 1,5 mL of 20% SDS (0,3% final) and ultra-pure water up to 100 mL total. 54. Resuspend carefully. 55. Incubate 1 h at 37 C with shaking 1400 rpm. 56. Add 27 mL of 20% Triton-X100 (1,8% final) and 173 mL 1.13 DpnII buffer (300 mL total). 57. Incubate 1 h at 37 C with shaking 1400 rpm. 58. Take 15 mL of mixture for integrity control and place it at 4 C until step 7 CRITICAL: Integrity control is important to check stability of DNA before restriction.     Repeat this step again. 103. Separate the beads on the magnet, discard the solution. 104. Leave the beads on the magnet for 5-10 min to air-dry the beads from the ethanol. 105. Remove the tube from the magnet. 106. Add 50 mL of 10 mM Tris-HCl pH 8.0 to the beads to elute the DNA. Mix gently by pipetting. 107. Incubate for 10 min at 22 C. 108. Place the tube on the magnet for 2-3 min. Transfer the solution to a fresh tube. 109. Prepare the mixture for end repair: 110. Incubate for 30 min at 20 C, then for 20 min at 75 C to deactivate the enzymes. 111. Keep at 4 C until the next step.
Note: The incubations 110-111 might be performed in a PCR machine.
Pause point: Samples might be kept at 4 C for 16-18 h.

Adapters preparation
CRITICAL: This step must be done in advance.
Note: It is possible to use Illumina kit adapters a. Prepare a mixture: b. Set the settings of PCR machine to perform the annealing procedure as follows: ramp at 0.5 C/s from 25 C to 97.5 C hold at 97.5 C for 2.5 min drop at 0.5 C/s from 97.5 C to 4 C keep at 4 C CRITICAL: Keep adapters at À20 C. 149. Incubate the beads solution at 98 C for 10 min to remove the DNA from the beads. The incubation might be performed in a PCR machine. In that case, set the program: 98 C-10 min followed by 4 C -N.
CRITICAL: This step helps separate beads and leave just DNA in the solution. It's critical because beads prevent fluorescence during Real-Time PCR 150. Place the tube on the magnet to separate the beads. Transfer the supernatant (DNA solution) to a new tube. 151. Set up the Real-Time PCR to determine the optimal cycle for final amplification.
The thermal cycle as follows: The optimal cycle is about 1 / 3 of the maximum fluorescence intensity. We chose 7 cycles.
152. Perform the PCR reactions on the remaining DNA template with the chosen number of cycles and the same conditions. We performed 3 reactions with 5 mL of DNA template for each one.

EXPECTED OUTCOMES
The protocol describes how to prepare both Hi-C and ATAC-seq libraries from fixed cortex tissue. The quality control of our libraries was performed by the GENEWIZ company. Figure 5 represents the library size distribution for ATAC-seq (A) and Hi-C (B). The ATAC-seq fragment size distribution has an expected profile. The first peak corresponds to the nucleosome-free library fragments, the second peak -mono-nucleosome fragments, the third peak -di-nucleosome fragments, the last peak -tri-nucleosome fragments. The average size of the ATAC-seq library fragments and Hi-C library is 497 bp and 557 bp, respectively.
ATACseq libraries were deep sequenced on the Illumina HiSeq by GENEWIZ (USA; www.GENEWIZ. com), resulting in 29-40 million 150-nt paired-end reads per sample. Sequencing reads were processed with the ATAC-seq nextflow pipeline (https://nf-co.re/atacseq) (Ewels et al., 2020) with default parameters, except for the peak calling option. The peaks were calculated by MACS2 (Zhang et al., 2008) with a narrow peak option and an initial threshold q-value of 0.05 as the cut-off. Reads

OPEN ACCESS
were aligned on mm10 genome assembly. The number of mapped reads as well as the number of mitochondrial reads for each replicate is indicated in the Table 1 below. As expected, the number of mitochondrial reads is low (0.48%-0.86%).
The distribution of fragment sizes was plotted with Picard (CollectInsertSizeMetrics) inside the nf-core ATAC-seq pipeline ( Figure 6). The fragment size distribution plot indicates a good quality of ATACseq data. The first peak corresponds to the nucleosome free region, the second -mononucleosome.
Hi-C libraries were deep sequenced on the Illumina NovaSeq by GENEWIZ (USA; www.GENEWIZ. com), resulting in 230.8-234.3 million 150-nt paired-end reads per sample. Sequencing reads were processed with the distiller-nf pipeline (https://github.com/open2c/distiller-nf). Reads were mapped on mm10 genome assembly with the default settings and with an option MAPQ30 filter. The number of uniquely mapped reads is indicated in the Table 2 below.

LIMITATIONS
Materials: In our protocol, we do not recommend substituting some reagents by other providers such asQubit ds; DNA BR Assay Kit; MyOne Strepavidin C1 beads; AMPure XP beads, Illumina Target DNA TDE1 Enzyme and Buffer Small kit, OptiPrepä Density Gradient Medium.  Isolation of Nuclei and FACS Compared to whole cells, nuclei are generally more fragile and the possible loss of nuclei during the isolation procedure can be expected. Moreover, sorting also could be limited due to the number of samples to be processed in a short period of time.

TROUBLESHOOTING Problem 1
The poor yield of nuclei after the iodixanol gradient (Isolation of nuclei from mice cortices) (step 12)

Potential solution
Make sure that the homogenate before the iodixanol gradient step does not contain any pieces of tissue. After the iodixanol gradient, take an interphase with nuclei carefully. The pipette tip position must be adjusted with the layer to maximize nuclei collection.

Problem 2
No interphase with nuclei observed after the iodixanol gradient (Isolation of nuclei from mice cortices) (step 12) Carefully add the layer of nuclei on top of the gradient. For this step it is crucial to use a bucket rotor centrifuge instead of a fixed-angle rotor centrifuge.

Potential solution
Optimization of nuclei number for transposition step is critical and directly impacts the library quality. We recommend calibrating the number of nuclei for your experiments. Using a large number of nuclei can result in incomplete transposition and bigger DNA fragments, while using a tiny number of nuclei can lead to over-transposition.
Problem 4 ATAC-seq fragment length distribution is small, around 200-300 bp (ATAC-seq) (step 29) Optimization of the incubation time with transposase is required. In most cases it indicates that the transposase concentration is too high compared to nuclei number.
It can rarely happen in case of sample contamination. We recommend performing the sorting of nuclei in sterile conditions.

Potential solution
Optimization of reverse-crosslinking reaction is required. Increasing the incubation time with proteinase K could be recommended.