GSM4984746: Brd4 dep Micro-C; Mus musculus; OTHER
ChIP-seq: Cells were cross-linked for 10 min at room temperature with 1% methanol-free paraformaldehyde in PBS and quenched with 0.125 M glycine. Cells were scraped and resuspended in cold cell lysis buffer (5 mM PIPES at pH 8.0, 85 mM KCl, 0.5% NP-40) and incubated for 10 min on ice. After centrifuging, nuclear pellets were resuspended in at least 6 vol of sonication buffer (50 mM Tris-HCl at pH 8.1, 10 mM EDTA, 0.1% SDS), incubated for 10 min on ice, and sonicated using the Covaris S220 sonicator to obtain an average fragment length of ∼500 bp examined by electrophoresis. Sonicated chromatin was diluted in RIPA buffer, aliquoted, and incubated with Protein G Dynabeads that were prebound with individual antibodies for at least 1 h at room temperature. Immunoprecipitation was performed overnight at 4°C with 2–4 µg of antibodies. Ten percent of the chromatin was saved as input. Immunoprecipitated and input DNA was introduced with sequencing adaptors as described previously (Schmidl et al., 2015), washed extensively with RIPA buffer (low salt and high salt) and LiCl buffer, digested with proteinase K and RNase A, reverse-cross-linked at 60oC overnight followed by purification with the AmpureX beads. We also added the spike-in chromatin (active motif, 53083) and spike-in antibody (active motif, 61686) following the manufacturer's instructions. Micro-C: 5M ESC cells were resuspended by trypsin, fixed by freshly made 1% formaldehyde at room temperature for 10 min and quenched by adding Tris buffer (pH = 7.5) to the final 0.75 M at room temperature. Fixed cells were washed twice with 1X PBS and then were subjected to the second crosslinking reaction by 3 mM disuccinimidyl glutarate (DSG) (Thermo Fisher # 20593)  for 45 min at room temperature. The DSG solution was freshly made at a 300-mM concentration in DMSO and diluted to 3 mM in 1X PBS before use. The crosslinking reaction was quenched by 0.75 M Tris buffer and washed twice with 1X PBS. Crosslinked cells were snap-frozen in liquid nitrogen and stored at −80°C.  We performed chromatin fragmentation by the micrococcal nuclease (MNase) and optimize the MNase digestion level to have about 80 to 90% of mononucleosomes and 10 to 20% of di-nucleosomes. We typically use ∼20 units MNase for 1 million of JM8.N4 mESCs. Intact nuclei were extracted by treating cells with Micro-C Buffer #1 (50 mM NaCl, 10 mM Tris-HCl pH = 7.5, 5 mM MgCl2, 1M CaCl2, 0.2% NP-40, 1x Protease Inhibitor Cocktail) for 20 min on ice. Chromatin was digested with a pre-titrated MNase concentration (Worthington Biochem #LS004798) at 37°C for 10 min. MNase digestion was stopped by adding 4 mM EGTA and completely inactivated by incubating at 65°C for 10 min. Digested chromatin was washed twice with ice-cold Micro-C Buffer #2 (50 mM NaCl, 10 mM Tris-HCl pH = 7.5, 10 mM MgCl2).MNase digested chromatin was subjected to multiple steps of biochemical enzyme reactions to generate compatible ends for ligation of adapters for sequencing. Chromatin was incubated with T4 PNK (New England BioLabs # M0201)  in Micro-C end-repair buffer (50 mM NaCl, 10 mM Tris-HCl pH = 7.5, 10 mM MgCl2, 100 ug/mL BSA, 2 mM ATP, 5 mM DTT) at 37°C for 15 mi. We then incubate chromatin with Klenow Fragment in the Micro-C end-repair buffer with no dNTPs at 37°C for 15 min. The blunting and labeling reaction was triggered upon adding biotin-dATP (Jena Bioscience # NU-835-BIO14), biotin-dCTP (Jena Bioscience # NU-809-BIOX), dGTP, and dTTP to a final concentration of 66 mM each at room temperature for 45 min. The reaction was inactivated by adding 30 mM EDTA and incubating at 65°C for 20 min. Biotin-labeled chromatin was washed once by ice-cold Micro-C Buffer #3 (50 mM Tris-HCl pH = 7.5, 10 mM MgCl2). The crosslinked nucleosomes were then ligated by T4 DNA Ligase (New England BioLabs #M0202) in 500 μL solution at room temperature for at least 2 h, followed by exonuclease III (New England BioLabs #M0206) treatment to remove biotin-dNTPs on un-ligated ends by incubating ligated chromatin at 37°C for at least 15 min.To specifically extract the ligated di-nucleosomal DNA, the deproteinized chromatin was purified and separated on a low-melting agarose gel (Lonza #50081). A band at the size of 250 to 400 bp corresponding to the ligated dimers was gel-extracted for library preparation. The purified DNA with biotin-dNTPs was captured by Dynabeads® MyOne Streptavidin C1 (Thermo Fisher Scientific #65001). Standard Illumina library preparation protocol including end-repair, A-tailing, and adaptor ligation was performed on beads with the NEBnext Ultra II kit (New England BioLabs # E7645). An optimal PCR cycle for final library amplification was determined by quantification PCR (KAPA Biosystems #KK4602), typically between 5–10 cycles for the input cell number from 5M. The sequencing library was amplified by Kapa HiFi PCR enzyme (KAPA Biosystems #KK2601) with the lowest possible cycles to reduce PCR duplicates. RNA-seq: Total RNA was isolated according to the manufacturer's instruction, quantified by Nanodrop (Thermo Fisher Scientific) and diluted to 1 ng/µl in nuclease-free water.  A total of 1 ng of RNA was added to 2.5 µl of cell lysis buffer (nuclease-free water with 0.2 % v/v Triton X-100 and 0.1 U/µl RNase inhibitor), and subjected to cDNA synthesis and amplification as described before (Cembrowski et al., 2018). Libraries were prepared using a modified Nextera XT DNA protocol (Illumina, San Diego, CA) where 5 µM P5NEXTPT5 was substituted for the i5 primers in the kit. Libraries were quantified by qPCR (Roche), normalized to 2 nM, then pooled and sequenced on a NextSeq550 flowcell with 25 bases in read 1, 8 bases in the i7 index read, and 50 bases in read 2. The control library phiX (Illumina) was spiked in at a final concentration of 15% to improve color balance in read 1. ATAC-seq: we performed ATAC-seq on 50,000 cells which were trypsinized, washed with PBS on ice, and resuspended in the reaction mix (25 μL of 2× TD buffer, 2.5 μL of transposase, 22.5 μL of nuclease-free H2O) for 30 min at 37°C. Following transposition, genomic DNA was purified and amplified by 10-14 cycles of PCR.Both ATAC-seq samples were sequenced in Illumina HiSeq 2500 or Nextseq with 150cycle paired-end sequencing