GSM8510255: HCEC_WT_Input; Homo sapiens; ChIP-Seq
Cells were crosslinked by addition of formaldehyde to a final concentration of 1%, and incubation for 10 min at RT. Fixation was stopped by addition of glycine to a final concentration of 0.125 M. Cells were washed in PBS, and harvested in SDS buffer (50 mM Tris pH 8.1, 0.5% SDS, 100 mM NaCl, 5 mM EDTA and protease inhibitors). Then, cells were pelleted down and resuspended in ice-cold IP buffer (1 vol. SDS buffer, 0,5 vol. Triton Dilution Buffer [100mM Tris-HCl ph 8.6, 100mM NaCl, 5mM EDTA, 5% Triton X-100] and protease inhibitors). Samples were then sonicated to an average length of 500-200 base pairs, using a Bioruptor Pico sonifier. The volume of the samples was adjusted with IP buffer to reach ~1ml per immunoprecipitation (9ug of DNA per IP sample). The sheared chromatin was blocked with Sepharose Protein-A beads. Next, a volume equal to 5% of the lysate used for each IP was removed, to be used later as the input samples. The rest of the lysates were incubated with 4ug primary antibody against H3K27ac (Abcam, Ab4729)followed by overnight incubation at 4C on a rotating wheel. The next day, lysates were centrifuged for 20 minutes at full speed, and the supernatants transferred to clean Eppendorf tubes pre-loaded with Blocked protein A beads followed by incubation for 3 hours at 4C on a rotating wheel. The beads were centrifuged and washed 3 times in Mixed Micelle Washing Buffer (150mM NaCl, 20mM Tris-HCl pH 8.1, 5mM EDTA, 5,2% w/v sucrose, 1% Triton X-100, 0,2% SDS), 3 times in Buffer 500 (0,1% deoxycholic acid, 1mM EDTA, 50mM HEPES, 500mM NaCl, 1% Triton X-100), 3 times in LiCl/detergent solution Buffer (0,5% deoxycholic acid, 1mM EDTA, 250mM LiCl, 0,5% NP-40, 10mM Tris-HCl pH 8) and once more in TE, before final resuspension in TE with 2% SDS and overnight incubation at 65C for decrosslinking. Finally, the beads were discarded by centrifugation and the supernatants moved to clean Eppendorf tubes, followed by DNA purification using a Qiaquick PCR Purification kit (Qiagen 28106). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit 96 (New England Biolabs BE7645L) according to manufacturer's instructions. Adapter ligation was carried out using the NEBNext Multiplex Oligos for Illumina (96 Dual Index Pairs) according to manufacturer's instructions. The quality and fragment size distributions of the purified libraries was assessed by a 4200 TapeStation Instrument (Agilent Technologies). Libraries were sequenced on a NovaSeq 6000 (Illumina) to an average read depth of 30M.