SO1B NTC 1512 + Break
Cells (5  10) were crosslinked in serum-free medium with 1% formaldehyde (Thermo Scientific, Cat#28908) for 10 min at room temperature, quenched with 0.125 M glycine for 5 min, and washed three times with ice-cold D-PBS. Pellets were either processed immediately or flash frozen at 80C.For lysis, pellets were resuspended in 1 mL LB1 (50 mM HEPESKOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100) with protease inhibitors (Fisher Scientific, Cat#PI78429) and incubated for 10 min at 4C with rotation. Cells were centrifuged (2000  g, 3 min, 4C), and pellets were resuspended in 1 mL LB2 (10 mM TrisHCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) with protease inhibitors, incubated for 5 min at 4C with rotation, and centrifuged again (2000  g, 3 min, 4C). Pellets were then resuspended in 0.5 mL LB3 (10 mM TrisHCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine) with protease inhibitors.Chromatin was sheared in LB3 using a Qsonica Cup Horn Sonicator (15 s on/45 s off, total on-time 6 min, 2550% amplitude) to achieve 200600 bp fragments. 25 L of each sonicated sample was collected for a sonication size check. Lysates were clarified by centrifugation (21,100  g, 10 min, 4C), adjusted to 1 mL with LB3 containing 1% Triton X-100, and 5% was reserved as input. Immunoprecipitations were performed overnight at 4C with 50 L Dynabeads Protein A or G (Invitrogen) pre-bound to 510 g antibody.Cleared lysates were adjusted to 1 mL with LB3 containing 1% Triton X-100, and 5% was reserved as input. Immunoprecipitations were performed overnight at 4C with 50 L Dynabeads Protein A or G (Invitrogen) pre-bound to 510 g of antibody. Beads were washed five times with ChIP wash buffer (50 mM HEPESKOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% IGEPAL CA-630, 0.7% sodium deoxycholate) and once with TBS (20 mM TrisHCl pH 8.0, 150 mM NaCl).The following day, immunoprecipitated samples were washed five times with ice-cold ChIP wash buffer (50 mM HEPESKOH, pH 7.5; 500 mM LiCl; 1 mM EDTA; 1% IGEPAL CA-630; 0.7% sodium deoxycholate) followed by a single wash with ice-cold TBS (20 mM TrisHCl, pH 8.0; 150 mM NaCl). Bound chromatin was then eluted twice at 65C with shaking at 1200 rpm for 15 min per elution using 60 L of ChIP elution buffer (0.1 M NaHCO, 1% SDS) for each round. To reverse proteinDNA crosslinks, the combined eluates were incubated overnight at 65C with shaking (1200 rpm) in the presence of 200 mM NaCl and 2 L RNase A (10 mg/mL). The following day, 2 L Proteinase K (20 mg/mL) was added, and samples were incubated for 1 h at 65C with shaking (1200 rpm). DNA was then purified using the MinElute PCR Purification Kit (QIAGEN, Cat#28004) and analyzed for enrichment by qPCR.