GSM5395715: Flavopiridol-ChIP-Rx-RPB1-DMSO; Homo sapiens; ChIP-Seq
Total RNA was extracted using Trizol. G4 ChIP-seq was performed according to a previously published protocol (Hansel-Hertsch et al.2018) with minor modifications. After fixation and chromatin shearing, 0.25% Triton X-100 was added to the chromatin and centrifuged at 20,000 g for 10 minutes to remove the insoluble debris.12.5 μl chromatin was immunoprecipitated with 800 ng BG4 antibodies and pre-blocked 5μl Anti-DYKDDDDK affinity beads (Smart-Lifesciences) sequentially. The beads were washed with 200 μl wash buffer (10 mM Tris-HCl at pH 7.4, 100 mM KCl, 0.1% Tween-20) at a 37°C rotating incubator (1,400 rpm) for 10 minutes. The captured DNA was eluted and reverse-crosslinked with 75 μl TE buffer and 1 μl 200 mg/mL proteinase K by incubation under rotation (1,400 rpm) for 1 hour at 37°C and then for 2 hours at 65°C. The DNA was further purified with phenol-chloroform extraction and ethanol precipitation. The G4 ChIP-seq libraries were prepared with the NEBNext ultra II DNA library prep kit for Illumina. The libraries were analyzed on an Agilent bioanalyzer and sequenced on a NovaSeq 6000. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed as previously described (Liang et al., 2015). Briefly, 1×107 cells were fixed with 1% paraformaldehyde in PBS for 10 minutes and then quenched with 0.125 M glycine for 5 minutes at room temperature. Fixed cells were sequentially lysed with 10 mL lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) and 10 mL lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors). The cells were then resuspended in 300 μl lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1× protease inhibitors), and were sheared using the Diagenode Bruptor Plus with the high power mode for 25 cycles (sonication cycle: 30 seconds ON, 30 seconds OFF). After sonication, 1% Triton X-100 was added to the chromatin and centrifuged at 20,000 g for 10 minutes to remove the insoluble debris. The chromatin was immunoprecipitated with 10 μg individual antibodies and 15 μl pre-blocked Protein A/G beads (Smart-Lifesciences). The beads were washed three times with 1 mL lysis buffer 3, and three times with wash buffer (50 mM HEPES-KOH, pH 7.5, 300 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate). The immunoprecipitated DNA was eluted with 200 μl elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1.0% SDS, 200 μg/mL proteinase K) at 55°C overnight, and purified by phenol-chloroform extraction and ethanol precipitation. Library preparation was done using the NEBNext ultra II DNA library prep kit for Illumina, followed by sequencing on a NovaSeq 6000. sonicated human chromatin was spiked-in with 10%-30% mouse chromatin toquantitatively normalize across experiments. Cleavage Under Targets and Tagmentation (CUT&Tag) was performed as described before (Kaya-Okur et al. 2019; Wang et al. 2019) with some modifications. Briefly, half million cells were washed twice in 1.5 mL wash buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) by gentle pipetting. 10 μL concanavalin A coated magnetic beads (Smart-Lifesciences) were activated, and added to these cells with a 10 minutes' incubation at room temperature. The supernatant was removed and bead-bound cells were resuspended in 100 μL dig-wash buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors,0.05% digitonin, 0.01% NP-40) containing 2mM EDTA and a 1:10 dilution of the 0.1 mg/mL BG4 primary antibody. Primary antibody incubation was performed on a rotating platform overnight at 4°C and then the liquid was removed. The Anti-Flag M2 antibody (Sigma-Aldrich) was diluted to 1:100 in 100 μL of dig-wash buffer with 2 mM EDTA, and was incubated with the cells at room temperature for 30 minutes followed by removing the liquid. The rabbit anti-mouse IgG antibody was diluted 100 folds and incubated with the cells for another 1 hour and washed with dig-wash buffer three times. A 1:200 dilution of pA-Tn5 adapter complex (~0.2 μM) was prepared in dig-300 buffer (0.05% digitonin, 20 mM HEPES at pH 7.5, 300 mM KCl, 0.5 mM spermidine, 1× protease 36inhibitors, 0.01% NP-40). 100 μL pA-Tn5 adapter complex was added to the cells with gentle vertexing, and was incubated at room temperature for 1 hour. Cells were washed three times in 200 μL dig-300 buffer to remove unbound pA-Tn5 proteins. Next, bead-bound cells were resuspended in 40 μL tagmentation buffer (10 mM TAPS-NaOH at pH 8.5, 10 mM MgCl 2 , 7.5% DMF) and incubated at 37°C for 1 hour. To stop tagmentation reaction, 1.5 μL 0.5 M EDTA, 0.5 μL 10% SDS and 0.5 μL 20 mg/mL proteinase K were added and incubated at 55°C for 1 hour and then for 15 minutes at 75°C. The DNA was extracted with Sera-Mag carboxylate-modified magnetic beads (GE Healthcare) for library preparation.21 μL DNA was mixed with a universal i5 and a uniquely barcoded i7 primer, and amplified with NEB Q5 high-fidelity 2× master mix. The libraries were purified with 1.1× volume of Sera-Mag carboxylate-modified magnetic beads, and subjected to LabChip DNA analysis and Illumina sequencing. ATAC-seq was performed as previously described (Corces et al. 2017). Briefly, 2.5×10 4 cells were washed with cold PBS and centrifuged at 600 g for 5 minutes. The cells were resuspended with 50μL cold lysis buffer (10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , 0.1% NP-40, 0.1% Tween-20 and 0.02% digitonin) and incubated on ice for 3 minutes. The cells were furthered added with 200 μL cold resuspension buffer (10 mM Tris-HCl at pH 7.5, 10 mM NaCl and 3 mM MgCl 2 ) and centrifuged at 600 g for 5 minutes. The pellet was resuspended with 12.5 μL resuspension buffer. 10 μL 5× tagmentation buffer (50 mM TAPS-NaOH at pH 8.5 and 25 mM MgCl 2 ), 16.5 μL PBS, 0.5 μL 10% Tween-20, 0.5 μL 1% digitonin, 0.75 μL 37.5 μM pA-Tn5 transposome and 9.5 μLnuclease-free H2O were mixed with 12.5 μL cell suspension. The transposition reaction was performed at 37°C for 60 minutes with mixing at 1,000 rpm. The reaction was stopped by adding 1.5μL 0.5 M EDTA and 0.5 μL 10% SDS, and digested with 1 μL 20 mg/mL proteinase K (Roche) at 65°C for 6 hours. The DNA was further purified with 1× Sera-Mag carboxylate-modified magnetic beads and eluted with 20 μL H 2 O with 0.1% Tween-20. The purified transposed DNA was amplified with NEBNext high-fidelity 2× PCR master mix, Ad1_noMX primer and Ad2.indexing primers. The DNA libraries were size-selected with Sera-Mag carboxylate-modified magnetic beads and sequenced on the NovaSeq 6000 platform. KAS-seq was performed as reported (Wu et al. 2020a) with minor modifications. Briefly, after drug treatment, HEK293T cells were labeled with 2.5 mM N 3 -kethoxal for 10 minutes. The cells were collected for genomic DNA purification. The extracted genomic DNA was biotinylated with DBCO-PEG 4 -biotin (Sigma-Aldrich) and sonicated with a Diagenode Bruptor Plus with a high power mode for 40 cycles (sonication cycle: 30 seconds ON, 30 seconds OFF). The fragmented N 3 -kethoxal- modified DNA was enriched with the Dynabeads Myone Streptavidin C1 beads (Thermo Fisher) and eluted with nuclease-free water at 95 ̊C for 15 minutes. The elutes were then repaired with the phi29 DNA polymerase (NEB) and random hexamers, and purified with the Sera-Mag carboxylate-modified magnetic beads. The libraries were prepared with the NEBNext ultra II DNA library prep kit. Indexed libraries were pooled and sequenced on an Illumina platform. TT-seq was performed as described previously (Schwalb et al. 2016; Liang et al. 2018) with minor modifications. 1×10 7 cells were labeled with 400 μM 4-thiouridine (4sU, Sigma-Aldrich) in a CO 2 incubator at 37°C for 10 minutes and were quickly lysed with 4 mL Trizol (Invitrogen). RN