title: GSM4773765: EPS-ATACseq-rep1; Mus musculus; ATAC-seq
library_construction_protocol: For RNA-seq, Total RNAs were extracted with TRIzol. For quantitative PCR, cDNAs were synthesized with ReverTra Ace (Toyobo) and oligo dT (Takara), and then analyzed by qPCR with SYBR green mixture (Genstar) and performed on a CFX Real-Time System (Bio-Rad). For ATAC-seq, a total of 50,000 cells were washed once with cold PBS and resuspended in 50 μl lysis buffer. The suspension of nuclei was then centrifuged for 10 min at 500 g at 4°C, followed by the addition of 50 μl transposition reaction mix (10 μl TD buffer, 5 μl Tn5 transposase and 35 μl nuclease-free H2O). DNA was purified using a MinElute Kit (QIAGEN). The BL-Hi-C libraries were performed as previous described. Briefly, the cells were treated with 1% formaldehyde for 10 min at RT and the crosslink was quenched by adding 2.5 M glycine. Then, the cells were treated with 0.1% SDS FA lysis buffer and then with 1% SDS FA lysis buffer. After that, the genome was digested with restriction enzyme HaeIII into fragments with blunt-ends, which were treated with adenine and ligated with bridge linker containing biotin at 16°C for 4 hr. The unligated DNA fragments were digested with DNA exonuclease. Next, the cells were treated with SDS and proteinase K to digest proteins, and the DNA was purified using phenol-chloroform extraction with ethanol precipitation. Then, the DNA was fragmented into 300 bp on average by sonication and the biotin-labeled DNA fragments were pulled down with streptavidin coated M280 beads. ChIP experiments were performed as previously described. Briefly, 1 × 107 cells were crosslinked with 1% formaldehyde at room temperature for 10 min. Then the reaction was stopped by adding glycine (final concentration, 0.125 M). Crosslinked cells were lysed in ChIP SDS lysis buffer（1% SDS, 10mM EDTA, 50mM Tris-HCl (pH 8.0)) containing 1 ×  protease inhibitor cocktail and PMSF, then sonicated to achieve a chromatin sized of 200-400 bp. After sonication, the supernatant was diluted with IP buffer and then co-incubated with antibodies–dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Immune complexes were washed with the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)) and TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Antibody-bound chromatin was reverse-crosslinked, and the ChIPed DNA samples were purified for ChIP-Seq library generation. For WGBS-seq, DNA was isolated using standard phenol/chloroform extraction and ethanol precipitation. C-T transformation of 1 μg genomic DNA and bisufile treatment were performed by using EZ DNA Methylation Gold™ Kit. Then modified DNA was treated as below: DNA fragment, end repair, A-tailing, ligate methylated adapter. And the target DNA samples were selected and treated with bisufile. RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina (Vazyme). ATAC libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme). BL-Hi-C libraries were constructed into standard illumina libraries. ChIP-seq libraries were constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2. WGBS libraries were constructed into standard illumina libraries.