GSM5124338: SC cells-H3K4me1-ChIP-seq; Homo sapiens; ChIP-Seq
For RNA-seq, RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries. For ATAC-seq, ATAC-seq assay was performed as previously described (Corces et al., 2017) with modifications. Briefly, SC cells were pretreated with DNase I (Worthington, LS002007) to digest DNA from dead cells and to remove free-floating DNA. The cells were counted with Crystal Violet (Sigma-Aldrich, C0775), and ~50,000 cells (for each sample) were resuspended in 1 ml cold ATAC-Resuspension Buffer (RSB; 3 mM MgCl2 [Sigma-Aldrich, M2670], 10 mM Tris-HCl pH 7.5 [ThermoFisher, 15567027], and 10 mM NaCl [Sigma-Aldrich, S7653] in sterile H2O). After centrifugation, the supernatant was discarded, and the cell pellets were resuspended in 50 μl cold RSB containing 0.1% Tween-20 (Sigma-Aldrich, P1379), 0.1% NP-40 (ThermoFisher, FNN0021) and 0.01% Digitonin (Sigma-Aldrich, D141). After lysis, 1 ml RSB containing 0.1% Tween-20 was added, and the tube was inverted several times to mix. After centrifugation, the supernatant was removed, and the nuclei were resuspended in 50 μl Transposition Mix (Vazyme, TD501). Then, the transposition reaction was incubated at 37°C for 25 min with shaking at 1,000 rpm. The reaction products were purified with the QIAquick PCR Purification Kit (QIAGEN, 28106). The ATAC-seq library was prepared using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) and TruePrep Index Kit V2 for Illumina (Vazyme, TD202) according to the manufacturer's instructions. The ATAC-seq analysis sequences with adaptor and low quality were trimmed for all 150-bp pair-end ATAC-seq reads. The clean reads were aligned to the human reference genome (GRCh37/hg19) using Bowtie2 (v2.2.1) (Langmead and Salzberg, 2012) in the default parameters. All the samples were separately processed, and duplicate reads were removed. The ATAC-seq peak regions of each sample were called using MACS2 (v2.1.1) (Zhang et al., 2008) in the default parameters. The results were analyzed and visualized in IGV (v2.8.6) (Thorvaldsdottir et al., 2013). 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). For 4C-seq, 4C assays were conducted as previously represented (Splinter et al., 2012; van de Werken et al., 2012b) with modifications. Briefly, SC cells were counted, and ~1 × 107 cells were used for each 4C experiment. The cells were cross-linked with 1.5% formaldehyde, quenched with 0.125 M glycine, lysed with cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, and 1× Protease Inhibitors), digested with DpnII (NEB, R0543M) for 4 h at 37°C while shaking at 900 rpm for more than 3 times until the digestion efficiency reached over 90%, and then ligated with T4 DNA ligase (NEB, M0202M) in a 7 ml reaction system after enzyme inactivation. After reversing the cross-links by adding Protein K (ThermoFisher, AM2548) for overnight at 65°C and RNase A for 45 min at 37°C, the ligation products were purified by Phenol/Chloroform/Isoamyl Alcohol (25:24:1) (ThermoFisher, 15593-049). The purified DNA samples were digested with NlaIII (NEB, R0125L) for the rs4728142-containing region (GRCh37/hg19 chr7: 128,573,714–128,574,084) or CviQI (NEB, R0639L) for the enhancer (GRCh37/hg19 chr7: 128,573,036–128,573,717) adjacent to the downstream rs4728142-containing region, and then ligated with T4 DNA ligase for overnight in a 14 ml reaction system. The ligation samples were purified by Phenol/Chloroform/Isoamyl Alcohol (25:24:1) and further purified utilizing the QIAquick PCR Purification Kit. The purified products were amplified by VAHTS HiFi Amplification Mix (Vazyme, N616) with 8 × 200 ng DNA. 4C-seq libraries were generated using the VAHTS Universal DNA Library Prep Kit for Illumina V3 according to the kit's instructions and then sequenced on a HiSeq X Ten platform. For data analysis, the quality control step was processed for the 4C-seq library using FastQC. The sequences with adaptor and low quality were discarded for further analysis. A Bioconductor package Basic4Cseq (Walter et al., 2014) was used to filter, analysis, and visualize the 4C-seq data. For RNA-seq, RNA libraries were prepared for sequencing using standard Illumina protocols.