GSM6600817: Krascells_ChIP_H3K27ac_Control_Clone1; Mus musculus; ChIP-Seq
ChIP-Seq libraries preparation ChIP-Seq experiments for H3K27ac, H3K4me3, H3K27me3were performed as described elsewhere (PMID: 28546318 and PMID: 32362324). Briefly, cells were fixed with 1% formaldehyde (ThermoFisher, #28906) at room temperature for 10 min. Cells were washed once again with 0.01%Triton-X-100 in PBS, pelleted, snap frozen and stored at -80ºC. Cells were thawed and lysed in a buffer containing 10 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5mM EGTA, 0.1% deoxcycholate, 0.5% sarkosyl, 1 3 protease inhibitor cocktail (SIGMA). Lysate was soni- cated using a Covaris for 12 cycles with the following setting: time, 60 s; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. One percent of sonicated lysate was kept as ChIP input for analysis. Immunoprecipitation mix consisting of Protein G Dynabeads (Invitrogen, #10003D) and 1 μg of H3K27ac (Active Motif, AB_2561016) or H3K4me3 (Millipore, 04-745). Beads were washed four times each with wash buffer I (20 mM tris-HCl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 2 mM EDTA), wash buffer II (10 mM tris-HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Na-deoxycholate, and 1 mM EDTA), tris-EDTA (TE) 0.2% Triton X-100, and TE 50 mM NaCl and then resuspended 25 μl of TT buffer (10 mM tris-HCl (pH 8.0) and 0.05% Tween 20, and sequencing libraries were prepared as following. ChIP libraries were prepared while bound to Dynabeads using a NEBNext Ultra II Library preparation kit (NEB) using half reactions. DNA was polished, poly(A)–tailed and ligated using dual UDI (IDT) or single (Bioo Scientific). Then crosslinks were revered by adding 33.5 μl of mix containing 18.4 μl nuclease free water, 4 μl 10% SDS, 3 μl  0.5 M EDTA, 1.6 μl 0.2M EGTA, 1 μl 10mg/ml Proteinase K (Biolabs, #P8107S), 1 μl 10mg/ml RNase A, 4.5 μl 5M NaCl by incubating at 55ºC for 1h followed by 75ºC for 30 min. Dynabeads were removed and libraries were cleaned by adding 3 μl of SpeadBeads in 125 μl of 20% PEG 8000/1.5M NaCl, washed by adding 120 μl 80% etOH, air dry and eluted in 12.5 μl of buffer containing 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. DNA was PCR-amplified for 15 cycles using NEBNext Ultra II PCR master mix using Solexa 1GA and Solexa 1GB primers. Libraries were size selected 200-500 bp by running in 10% TBE acrylamide gels (ThermoFisher, #EC62752BOX) and sequenced using a HiSeq 4000 or a NextSeq 500.   ATAC-seq library preparation Transposase reactions and sequencing libraries were generated as described previously (PMID: 24097267) 100,000 cells. Tagmented DNA was purified using Zymo ChIP Clean & Concentrate columns and PCR amplified for 10-12 cycles using barcoding primers. Libraries were size selected by gel excision to 175-225 bp using 10% TBE acrylamide gels (ThermoFisher, #EC62752BOX). RNA-Seq library preparation It was performed as described somewhere else (PMID: 28546318 and PMID: 32362324). Briefly, cells were lysed in Trizol. RNA and DNase treatment was carried out using Direct-zol RNA MicroPrep kit (Zymoresearch, #11-33MB). 1 μg total RNA was enriched in poly-A tailed RNA transcripts by double incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and fragmented for 9 min at 94ºC in 2X Superscript III first-strand buffer containing 10mM DTT (Invitrogen, #P2325). Reverse-transcription (RT) reaction was performed at 25ºC for 10 min followed by 50ºC for 50 min. RT product was purified with RNAClean XP (Beckman Coulter, #A63987). Libraries were ligated with dual UDI (IDT) or single (Bioo Scientific), PCR-amplified for 11-13 cycles, size-selected using one-sided 0.8× AMPure cleanup beads, quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and sequenced on a HiSeq 4000 or NextSeq 500 (Illumina).