ATAC-seq was performed as the following decription. Briefly, 50,000 cells were washed once with 50 μl of ice-cold PBS, then lysed with 50 μl of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin) by incubating on ice for 10 min. The pellet was washed once with 1 ml of washing buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, and 0.1% Tween-20). Tansposition reaction was performed in 50 μl of reaction mix containing 10 μl of 5× TTBL (Vazyme, TD501), 5 μl of TTE Mix V50 (Vazyme, TD501), 16.5 μl of PBS, 0.5 μl of 10% Tween-20, 0.5 μl of 1% digitonin, and 17.5 μl of PCR-grade water. The mixture was incubated at 37 ˚C for 30 min on a thermomixer with shaking at 1,000 rpm. Then, DNA was extracted by phenol-chloroform and amplified by KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2611) with barcoded primers (Vazyme, TD202). DNA fragments ranging from 200 bp to 1,000 bp were selected for deep sequencing. For ChIP-seq, Cells were harvested in PBS and cross-linked with 1% formaldehyde for 9 min at room temperature, cells prepared for RNA pol II were pre-cross-linked with 0.2 mM disuccinimidyl glutarate (DSG, ProtecoChem, c1104) for 30 min at room temperature. Reaction was terminated by adding glycine to a final concentration of 125 mM, incubated at room temperature for 5 min. After washing with PBS twice, cells were resuspended in 5 mL of Lysis Buffer 1 (50 mM HEPES, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25%Triton X-100), incubated on ice for 10 min. After centrifuge at 2,000 rpm at 4 ˚C for 5 min, cells were then resuspended in 5 mL of Lysis Buffer 2 (10 mM Tris-HCl, pH8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA), incubated at room temperature for 10 min. The lysate was centrifuged at 4,000 rpm for 5 min at 4 ˚C. The chromatin fraction was resuspended in 1 mL of Lysis Buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% sodium N-lauroylsarcosine, 1x proteinase inhibitor (Merck, 5892970001)). Chromatin was then sonicated into 200-400 bp fragments (Covaris M220), in assistance with 0.1% sodium dodecyl sulfate (SDS) or 0.5% SDS (for double cross-linked sample). Supernatant after centrifuging at 12,000 rpm for 10 min at 4 ˚C were flash freezed and kept in -80 ˚C refrigerator.    Keep 10% of chromatin as input, the fragments were immunoprecipitated overnight with antibodies against histone modifications or RNA pol II. Pre-washed Dynabeads protein A or G (ThermoFisher 10002D, or 10004D) was added to the Protein-antibody complexes, and incubated at 4 ˚C for 1 hour. The beads were sequentially washed with buffers as follows: FA-lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM HEPES, pH 7.9, 2 mM EDTA and 0.5 mM sodium deoxycholate), FA high salt buffer (500 mM NaCl, 1% Triton X-100, 50 mM HEPES, pH 7.9, 2 mM EDTA and 0.5% sodium deoxycholate) for twice, LiCl buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0), TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA) for twice. Each washing step was incubated for 5 min at 4 ˚C with rotation. Protein-DNA complexes were eluted by incubation with 250 µl of elution buffer (1% SDS, 100 mM NaHCO3, freshly made) for 15 min at 55 ˚C with periodic gentle vortex. This step was repeated once and eluted sample was combined.    Eluted DNA, as well as input, was reverse cross-linked from protein by adding 20 µl of 5M NaCl to the 500 µl of elution, incubated at 65 ˚C over 8 hours. After digested with RNase at 37 ˚C, proteinase K at 55 ˚C, DNA was extracted by phenol-chloroform method. The ChIP-seq libraries were constructed using NEBNext DNA Sample Prep Master Mix (NEB).DNA libraries were subjected to size-selection using AMPure XP beads (Beckman Coulter) and PCR amplification (18-20 cycles for ChIP-seq DNA libraries).
GEO Accession
GSM4377779