GSM9465179: HROhep03_siKDM6A_H3K27me3_rep3; Homo sapiens; ChIP-Seq
Fresh liver tissues (chopped into small pieces) and HROhep03 cells were crosslinked with 1% formaldehyde (ThermoFisher, 28906) in PBS for 10 minutes for histone modifications. For transcription factors (TFs), tissues/cells were double crosslinked with 2 mM disuccinimidyl glutarate (DSG) (ThermoFisher, 20593) for 30 minutes, followed by 1% formaldehyde for 10 minutes. The reaction was stopped with glycine at a final concentration of 0.125 M for 5 minutes. Liver pieces were disaggregated in ice-cold PBS with protease inhibitor using a Dounce Homogenizer, first with a loose pestle and then with a tight pestle (Fisher Science, FB56691). Nuclei were isolated using three lysis buffers: lysis buffer 1 (50 mM Hepes–KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630, and 0.25% Triton X-100), lysis buffer 2 (10 mM Tris–HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), and lysis buffer 3 (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, and 0.5% N-Lauroylsarcosine). The samples were then sonicated for 30 minutes (30 seconds ON/30 seconds OFF) using the Bioruptor Pico (Diagenode). For ChIP-seq sample preparation, the same ChIP protocol was followed, but the ChIP DNA was purified using the ChIP DNA Clean and Concentrator Capped Zymo-Spin I (Zymo Research) purification kit. Two to four ChIPs were pooled during the final purification step to obtain concentrated material. For library preparation and sequencing, 2–10 ng of ChIPed DNA was processed using the Rubicon ThruPLEX DNA-seq kit (TAKARA) following standard protocols, and sequenced on the NovaSeq X Plus Series (150PE reads, NOVOGENE, United Kingdom).