title: GSM9252903: Jurkat cell resting ChIP-seq H3K27me3 rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: 1 × 10^7 Jurkat cells were cross-linked with 1% formaldehyde (Sigma-Aldrich, F8775) in 10% FBS/phosphate-buffered saline (PBS) for 9 minutes at room temperature and subsequently quenched with 0.125 M glycine (Sigma-Aldrich, 50046). The cells were then collected and lysed twice using ice-cold lysis buffer, which consisted of 20 mM Tris-HCl (pH 7.5) (Thermo Fisher Scientific, 15567027), 2 mM EDTA (Thermo Fisher Scientific, 15575020), 1% Triton X-100 (Sigma-Aldrich, T8787), 0.1% SDS (Thermo Fisher Scientific, 15525017), 0.1% sodium deoxycholate (Sigma-Aldrich, D6750), and 1× protease inhibitors (Thermo Fisher Scientific, 78425) for 15 minutes with slow rotation at 4°C. The nuclear lysates were sonicated to obtain DNA fragments ranging from 200 to 500 bp using a Biorupter system (Diagenode, B01060001) at high energy, with a working time of 30 seconds and a resting time of 30 seconds, for a total of 30 cycles, until the solutions became clear. After removing insoluble debris, the lysates were incubated separately with specific antibodies against Rabbit IgG (1:100, Cell Signaling Technology, 7074), CTCF (1:100, Abcam, ab128873), ZNF384 (1:100, Novus Biologicals, NBP3-30592), H3K27me3 (1:100, Cell Signaling Technology, 9733), Rpb1 NTD (1:100, Cell Signaling Technology, 14958), H3K4me1 (1:100, Cell Signaling Technology, 5326), H3K4me3 (1:100, Cell Signaling Technology, 9751), or H3K27ac (1:100, Abcam, ab4729), and then purified using protein A-agarose beads (Millipore, 16-157). The beads were washed four times with TE buffer (10 mM Tris-HCl pH 8.0 [Thermo Fisher Scientific, 15568025] and 1 mM EDTA), TSE I buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), TSE II buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), and Buffer III (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 0.25 M LiCl [Sigma-Aldrich, L4408], and 1% sodium deoxycholate) before elution (20 mM Tris-HCl pH 7.5, 5 mM EDTA, 50 mM NaCl, and 1% SDS), reverse cross-linking, and subsequent DNA extraction. ChIP-seq libraries were generated using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607) according to the kit's instructions and were sequenced on a HiSeq X Ten platform (Illumina).