GSM8499403: Donor 1362, WT, YY1; Homo sapiens; ChIP-Seq
Briefly, 50mln cells per immunoprecipitation were collected and fixed with 1% formaldehyde for 15 min and then quenched with 0.25 M glycine for 5 min on ice. After 3 washes with 1X PBS, pellets were resuspended in 10 mL each of a series of two lysis buffers and resuspended in 1mL of the third lysis buffer before fragmentation in Covaris ME220 Ultrasonicator to generate chromatin fragments roughly 200–500 bp in size. Chromatin was centrifuged to clear debris and a 1:20 of this cleared chromatin was kept as standard input for comparison against immunoprecipitations. Chromatin was incubated rotating at 4°C 1h with the specific antibody and chromatin–antibody complexes were precipitated using 250 µL of Dynabeads Protein A at 4°C overnight. DNA was purified using Zymo ChIP DNA Clean & Concentrator Kit Libraries for sequencing were made using NEBNext Ultra II DNA Library Prep Kit