GSM6329648: FLAG-MESP1_ChIP_in_Dox+MESP1_CPC_rep2; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation (ChIP) and ChIP-seq were performed as described previously (Lan et al., 2007; Niu et al., 2021). Cells were cross-linked with 1% formaldehyde for 10 minutes, quenched with 0.125 M glycine for 10 minutes; then, the cell pellets were wash twice by cold PBS, and then resuspended in high salt Lysis buffer (50 mM HEPES/KOH, pH 7.5, 500 mM NaCl, 1% Trixon-X 100, 0.05% SDS, 10 mM EDTA, and proteinase inhibitors), and sonicated with Qsonica (Misonix) to shear the chromatin into 200- to 400-bp lengths. Chromatin was immunoprecipitated with the indicated antibodies overnight and MagPoly protein A/G magnetic beads (Smart, Changzhou, China) for 2 hours. Then the beads were washed 3 times of high salt lysis buffer, 3 times of low salt buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40, 0.5% Na-Deoxycholate), once of 50mM HEPES/KOH, pH 7.5, and DNA was eluted in elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) with Proteinase K (Invitrogen-Thermo Fisher, Waltham, MA) in 72℃ for 3 hours. DNA was isolated by MinElute PCR Purification Kit (QIAGEN 28006). RNA-seq: Total RNA was isolated using TRIzol (Life Technologies). Non-ribosomal RNA was isolated from 1 μg total RNA by using a TrueLib Poly (A) mRNA Magnetic Isolation Module (Excell Bio, Shanghai, China), and sequencing libraries were prepared with the TrueLib mRNA Library Prep Kit for Illumina (ExCell Bio, Shanghai, China). Hi-C: 1,000,000 Cells were cross-linked with 1% formaldehyde for 10 minutes, quenched with 0.2 M glycine for 5 minutes; then, the cell pellets were wash twice by ice cold PBS, and then resuspended in 300 μl of ice cold Hi-C lysis buffer (10 mM Tris-HCl 8.0, 10 mM NaCl, 0.2% NP40, cOmplete Ultra protease inhibitors) and incubated on ice for 15 min. Samples were then centrifuged and washed once with 500 μl of ice cold Hi-C lysis buffer. Nuclei were centrifuged (2,500 g for 5 min at 4℃) and permeabilized by resuspending them in 20 μl of 0.5% SDS and incubating at 62℃ for 10 min with shaking (1,000 rpm). SDS was quenched by adding 58 μl of nuclease-free water and 10 μl of 10% Triton X-100 and incubated at 37℃ for 15 min with shaking (1,000 rpm). Chromatin was digested by adding 100 U of DpnII and 10 μl of 10x DpnII buffer for 2 hours at 37℃ with rotation. DpnII was heat-inactivated at 62℃ for 20 min. The overhangs generated by the restriction enzyme were filled-in by adding a mix of  0.4 mM biotin-dATP (10 μl; PicoHeliex), 10 mM dCTP/dGTP/dTTP (0.5 μl of each dinucleotide), and 5 U/μl DNA polymerase I，Large（Klenow）Fragment (8 μl; New England Biolabs), and incubated for 90 min at 37℃ with rotation. DNA fragments were ligated in 10X T4 DNA ligase buffer (5 μl), 10mM dATP(10 μl), 10 mg/mL BSA (1.5 μl) and 400 U/μl T4 DNA ligase (1.5 μl; NEB) by incubating 8 hours at 20℃. Protein and RNA were digested by adding 5 μl of 20 mg/mL Proteinase K (Thermo Scientific), 10 mg/ml RNase A (2 μl ; Thermo Scientific), 20 μl of 10% SDS and 25 μl of nuclease-free water and incubated at 65℃ with shaking (1,000 rpm) for 8 hours. Phenol-Chloroform-Isoamyl alcohol (25:24:1; Solarbio) extracted DNA was resuspended in 110 μl of 10mM Tris pH 8.0 and incubated for 15 min at 37℃. Samples were sheared using a Qsonica sonicator (6 cycles, each 30 s, 80% duty). DNA size was selected with VAHTS DNA Clean Beads (Vazyme Biotech, Nanjing, China) to get 250 – 500 bp length of DNA fragments dissolved in 300 μl of 10mM Tris pH 8.0. Biotinylated DNA fragments were pulled down using Streptavidin MagPoly Beads (30 μl; Smart Lifesciences, Changzhou, China) supplemented with 300μl of 2X binding buffer (10mM Tris pH 7.5, 1mM EDTA, 2M NaCl) and incubated in 25℃ with rotation for 15 min. The biotinylated DNA fragments on streptavidin beads were washed twice with 600 μl of Tween Wash buffer (5mM Tris pH 7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20) incubated in 55℃ with shaking (800 rpm) for 2min. The Hi-C DNA libraries were prepared as directed by the protocol of VAHTS Universal DNA Library Prep Kit for Illumina (Vazyme Biotech, Nanjing, China). ChIP DNA libraries, input DNA libraries and HiC DNA libraries were prepared as directed by protocol of VAHTS Universal DNA Library Prep Kit for Illumina (Vazyme Biotech, Nanjing, China).