GSM6383108: K562 HS HSF1 rep 2; Homo sapiens; ChIP-Seq
Approximately 2x107 cells were crosslinked with 1% ethanol-free formaldehyde (Thermo) in serum-free DMEM/F12 media at 25C for 10min, quenched with 125mM Glycine, and lysed in buffer (1% Sodium-Dodecyl Sulfate (SDS), 50mM Tris (pH=8), 10mM EDTA, 1X PMSF and protease inhibitor cocktail) followed by disruption in the Covaris S2 sonicator for 6 minutes (with peak power = 140, Duty factor = 5, Cycles/burst = 200). Protein A and Protein G Dynabeads (Invitrogen) premixed at 1:1 ratio were used for pre-clearing for 1 hour at 4C. One percent of each precleared sample was taken as input, with at least two inputs taken for each sample and averaged for ChIP percent input calculation. Samples were incubated with 5g of primary antibodies in 2 ml of dilution buffer (1% Triton-X, 2mM EDTA, 20mM Tris pH=8.0, 150mM NaCl, 1X Phenylmethylsulfonyl fluoride (PMSF) and cOmplete protease inhibitor cocktail (Sigma)) with slow rotation overnight at 4C followed by washing DNA library was prepared from validated ChIP material using NEBNext Ultra II DNA library kit (New England Biolabs) protocol without size selection. Final PCR amplification of cDNA templates was done using TruSeq Illumina Small RNA PCR primers, 250 M dNTP mix, 1x HF buffer, 1M Betaine, 1xEvagreen dye (Biotium) and Phusion DNA polymerase.