GSM8508510: H3k4me3_TaDRIM-Seq-293T-rep2; Homo sapiens; OTHER
Approximately1×106- 1×107 crosslinked cells were used per replicate. Cell nuclei were isolated in 1 mL lysis buffer (10mM Tris pH 7.5, 10mM NaCl, 0.2% NP40, 1×protease inhibitors, 0.2 U/μl RiboLock RNase) (Thermo Fisher), and incubated for 10min on ice followed by centrifugation for 3min at 2500g and 4°C. The nuclei were washed twice with 300µl 1×wash buffer A (20mM HEPES pH 7.6, 150mM NaCl, 0.5mM Spermidine, 1×protease inhibitors, 0.2U/μl RiboLock RNase) (Thermo Fisher), and nuclei were collected by centrifugation for 3min at 2500g and 4 °C. Nuclei were then resuspended in Antibody buffer (495μl 1×wash buffer A, 5μl 5% digitonin, 2μl 0.5M EDTA, 1.6μl 30%BSA, 10μg antibody (H3K4me3, Abcam, ab8580; H3K9ac, Abcam, ab10812; CTCF, Abcam, ab188408), and incubated at 4°C for 2h with rotation. Nuclei were pelleted for 3 min at 2500g and washed five times with 300μl 1×wash buffer A, resuspended 500µl 1×transposase incubation buffer (20mM HEPES pH 7.6, 300mM NaCl, 0.5mM Spermidine, 0.01% digitonin, 1×protease inhibitors, 0.2U/μl RiboLock RNase) (Thermo Fisher), followed by adding 5μl TTE Mix, and incubated at 4°C for 1h with rotation. Nuclei were collected and washed five times with 300µl 1×transposase incubation buffer and resuspended in tagmentation buffer (500µl 1×transposase incubation buffer, 5µl 1M MgCl2), and incubated at 37°C for 1h. Next, 500µl of 40mM EDTA (Thermo Fisher) was added to stop the reaction. The isolated nuclei were washed twice with 500µl 1×PBS (Thermo Fisher) and then incubated in 200µl permeabilization buffer (190µl nuclease-free water, 10µl 10% SDS) (Thermo Fisher) at 62°C for 10min, and immediately quenched the SDS reaction by adding add 580μl of nuclease-free water and 100μl of 10% (vol/vol) Triton X-100 (Sigma-aldrich), and incubated at 37°C for 15min with rotation.The isolated nuclei were washed twice with 500µl 1×wash buffer B (20mM Tris-HCl, pH 7.5, 10mM MgCl2 and 0.2% Tween 20) and resuspended in 500µl 1× CutSmart buffer (NEB), followed by adding 1.6µl RNase If (NEB) with dilution tenfold in 1× PBS, and incubated at 37°C for 3min with rotation followed by incubation for 5min on ice. Nuclei were collected by centrifugation, then resuspended by 400µl PNK solution containing 1×T4 PNK reaction buffer, 1mM ATP, 1U/μl RiboLock RNase and 1U/μl T4 PNK (Thermo Fisher) at 37°C for 40min with rotation. The specific bridge linker was designed containing 5ʹ phosphorylation [5Phos] and Internal Biotin dT [ibiodT] chemical modifications. The forward strand is: 5′-[5APP]CCTCGATAC/iBIOdT/TGAGCTGAC-3′, and reverse strand is 5′-[5Phos]GTCAGC/iBIOdT/CAAGTATCGAG-3′. The 5′App ends of forward strand was treated by using a DNA 5′ Adenylation Kit (NEB). The isolated nuclei were washed twice with 500µl 1×wash buffer B and resuspended in 400µl of RNA ligation solution (1×T4 RNA ligase reaction buffer, 1U/μl RiboLock RNase, 10U/μl T4 RNA Ligase 2 truncated, 1.5pmol/μl bridge linker, 15% (w/v) PEG 8000, 0.1% Triton X-100) (NEB), and incubated overnight at 16 °C with rotation. The nuclei were pelleted by centrifugation, resuspended in 400 μl reverse transcription solution (1×MMLV buffer, 0.5mM dNTP, 8U/μl MMLV (RNase H-), 1U/μl RiboLock RNase) (Promega), and then incubated at 42 °C for 1 h. The isolated nuclei were washed five times with 500μl 1×wash buffer B, resuspended in T4 DNA ligation solution (1×DNA ligase reaction buffer with 10mM ATP, 4U/μl T4 DNA ligase, 0.1% (vol/vol) Triton X-100 and 0.1 mg/mLBSA) (NEB), incubated overnight at 16 °C with rotation. The isolated nuclei were resuspended in proteinase K solution (50 mM Tris-HCl, pH 7.5, 1% SDS, 100mM NaCl, 1mM EDTA and 1mg/mL proteinase K) at 65 °C for 3h with shaking at 800 rpm. RNA/DNA complex was extracted and dissolved in nuclease-free water (Thermo Fisher). The purified RNA/DNA complexes were incubated with gap-filling solution (10×NEBuffer r2.1,1mM dNTP and 0.18U/μl T4 DNA polymerase) (NEB) at room temperature for 40min to repair the pG-Tn5 transposition gap, followed by second-strand cDNA synthesis. DNA-cDNA complexes were converted to DNA library according to the TruePrep DNA Library Prep Kit for Illumina (Vazyme, cat. no. TD501). The fragments containing biotinylated bridge-linker were enriched by M280 beads (Thermo Fisher) before PCR, and enriched library DNA is size-selected by using AMPure XP beads (Beckman), and sequenced (2×150 bp) using Illumina Hiseq X-Ten (Annoroad Gene Technology).