For ChIP-seq, ChIP assays were performed as previously described (Li et al., 2017c) with modifications. Briefly, for each sample, ~1 × 107 SC cells were cross-linked with 1% formaldehyde (Sigma-Aldrich, F8775) in 10% FBS/phosphate-buffered saline (PBS) for 10 min at room temperature and quenched with 0.125 M glycine (Sigma-Aldrich, 50046). The cells were then collected and lysed twice with ice-cold lysis buffer (20 mM Tris-HCl pH 7.5 [ThermoFisher, 15567027], 2 mM EDTA [ThermoFisher, 15575020], 1% Triton X-100 [Sigma-Aldrich, T8787], 0.1% SDS [ThermoFisher, 15525017], 0.1% Sodium Deoxycholate [Sigma-Aldrich, D6750], and 1× Protease Inhibitors [ThermoFisher, 78425]) for 10 min with slow rotations at 4 °C. The nuclear lysates were sonicated to obtain DNA fragments of 200~500 bp using a Biorupter system (Diagenode, B01060001) by high energy, with a working time of 30 s and a resting time of 30 s, 30 cycles, until the solutions turned clear. After removing insoluble debris, the lysates were separately incubated with specific antibodies against Rabbit IgG (CST, 7074), ZBTB3 (Novus Biologicals, NBP1-82079), CTCF (Abcam, ab128873), RAD21 (Abcam, ab992), H3K4me1 (CST, 5326), H3K4me3 (CST, 9751), or H3K27ac (Abcam, ab4729), and then purified by protein A-agarose beads (Millipore, 16-157). The beads were washed 4 times by TE buffer (10 mM Tris-HCl pH 8.0 [ThermoFisher, 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 then sequenced on a HiSeq X Ten platform (Illumina). For data analysis, the sequences with adaptor and low quality were trimmed for all 150-bp pair-end reads. The clean reads were mapped against the human reference genome (GRCh37/hg19) using BWA (Li and Durbin, 2009). All the samples were separately processed, and the unique mapped reads were kept. The peaks were called by MACS2 (v2.1.1) (Zhang et al., 2008) using IgG as control. The results were analyzed and visualized in IGV (v2.8.6) (Thorvaldsdottir et al., 2013).