GSM7764480: Control, rep3; Mus musculus; ChIP-Seq
Nuclei were harvested by sequential lysis in lysis buffer one (LB1), two (LB2), and three (LB3) which were supplemented with protease inhibitor (ThermoFisher Scientfic Cat# 78429; 1X final concentration added right before use). LB1 consists of 50mM HEPES-KOH (pH 7.5, MilliporeSigma Cat# H4034 & P9333), 140mM NaCl (MilliporeSigma Cat# 7653), 1mM EDTA (pH 8.0, MilliporeSigma Cat# E6758), 10% glycerol (MilliporeSigma Cat# G5516), 0.5% Igepal CA-630 (MilliporeSigma Cat# I8896), 0.25% Triton X-100 (MilliporeSigma Cat# T8787) and KOH to adjust pH to 7.5. LB2 consists of 10mM Tri-HCl pH 8.0 (Quality Biological Cat# 351-007-101), 200 mM NaCl, 1mM EDTA pH 8.0, 0.5 mM EGTA and HCl to adjust pH to 8.0. LB3 consists of 10mM Tris-HCl (pH 8.0), 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Sodium deoxycholate (MilliporeSigma Cat# 30970), 0.5% N-lauroylsarcosine (MilliporeSigma Cat# L7414) and HCl to adjust pH to 8.0. Cells were re-suspended in LB1 and rotated for 10 minutes at four degrees Celsius. Then, cells were pelleted at 2000 rpm, re-suspended in LB2 and rotated for 5 minutes at four degrees Celsius. Then, cells were pelleted at 2000 rpm and re-suspended in LB3. Nuclei were then sonicated at 4 degrees Celsius using a Fisher 150E Sonic Dismembrator with settings: 50% amplitude, 30 s ON, 30 s OFF for 12 min total time. Following sonication, samples were pelleted to remove cellular debris at 20,000g for 10 minutes at four degrees. After IP, DNA was extracted from reverse crosslinked chromatin samples by incubation with RNaseA (Thermo Fisher Scientific Cat# EN0531) for 15 min at 37C; and proteinase K (Thermo Fisher Scientific Cat# AM2546) for 1h at 65C. DNA was subsequently purified using MinElute (Qiagen Cat# 28004) purification. DNA libraries were prepared and amplified using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs Cat# E7103L) and dual-indexed NEBNext Multiplex Oligos for Illumina (New England BioLabs Cat# E6440S). Final libraries were pooled, quantified with QuBit (Thermo), Bioanalyzer (Agilent) and qPCR (BioRad), then sequenced on a Novoseq SP 100 flow cell (Illumina) using paired-end 2x50bp