GSM7434990: ZR751_DMSO-IgG-rep1; Homo sapiens; ChIP-Seq
ChIPseq was performed as previously described (Zhou et al., 2022). Briefly, Dynabeads Protein G (Invitrogen, Cat. 10003D) were resuspended in PBS+0.5% BSA and incubated with 30 μg of antibodies recognizing Pol2 (CST, cat. 14958S) or Pol2 S2P (Abcam, ab5095) for 8h at 4°C. 3x 107 ZR75-1 cells treated with DMSO or PF-9363 for 24h, were crosslinked for 30min at RT in PBS with 1.5mM ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester) (EGS) (Thermo Fisher, Cat. 21565) followed by 15min with 1% formaldehyde (Thermo Scientific, Cat. 28908) and quenched with 0.2M glycine. Crosslinked cells were washed 2x with PBS, lysed and sonicated on Covaris M220 with PIP 75, DF 10, CPB 200 for total of 8min. Chromatin lysates were cleared by centrifugation and Triton X-100 was added to a final concentration of 1%. Lysates were incubated overnight at 4°C with Protein G Dynabeads conjugated to antibodies against Pol2 or Pol2 S2P. Beads were washed with low salt wash buffer, high salt wash buffer, LiCl buffer and TE with 50mM NaCl and DNA-protein complexes were eluted twice for 30 min at 65 °C with shaking. Buffers in all steps above were supplemented with protease inhibitors (Roche, Cat. 11697498001). Input sample was prepared by the same approach without immunoprecipitation. RNase (Thermo Scientific, Cat. FEREN0531) and proteinase K (Invitrogen, Cat. 25530049) treatments were performed at 37 °C for 1h and 55 °C for 2h, respectively, and samples were de-crosslinked overnight at 65 °C. DNA was purified using Qiagen QiaQuick PCR purification kit (Qiagen, Cat. 28106) and quantified with Qubit (Invitrogen, Cat. Q32854). Cut&Run was performed using CUTANA™ ChIC/CUT&RUN kit (EpiCypher, Cat. 14-1048) per manufacturer recommendations. Briefly, ZR-75-1 cells or MCF7 cells were fixed with 0.1% formaldehyde (Fisher Scientific, Cat. F79-500) for 1min and fixation was quenched with 125mM glycine (Fisher Scientific, cat. AAJ1640736). Fixed cells were washed with PBS (Gibco, Cat. 10010-031) and nuclei were isolated with nuclear extraction buffer (20mM HEPES, pH7.9, 10mM KaCl, 0.1% Triton X-100, 20% glycerol, 1mM MnCl2) and inspected under the microscope to confirm efficiency of nuclei extraction and their integrity. 5x105 nuclei were washed with wash buffer and immobilized to Concavalin-A beads, resuspended in 50 µl antibody buffer and incubated with 0.5 µg of the following antibodies: IgG (EpiCypher, Cat.13-0042k), H3K4me3 (EpiCypher, Cat.13-0041k), H3K9ac (EpiCypher, Cat.13-0033), H3K27ac (EpiCypher, Cat. 13-0045), H3K27me3 (Thermo-Fisher, MA5-11198) or H3K23ac (CST, Cat.14932S or Abcam, ab177275) overnight at 4°C. Beads were washed with Cell Permeabilization buffer and incubated with pAG-MNase for 10min at RT. After activation by CaCl2 beads were incubated for 2h at 4°C, Stop buffer was added together with 0.5ng of E. coli Spike-in DNA and incubated at 37°C for 10min to release Cut&Run fragments from the insoluble nuclear chromatin. DNA was then decrosslinked overnight at 55°C and purified using DNA Cleanup Columns from the kit. Wash buffer, Cell Permeabilization buffer and Antibody buffer were supplemented with 0.05% SDS (Invitrogen, Cat. 15553027) and 1%Triton X-100 (Sigma, Cat. 93443-500ML). Libraries were prepared using the NEBNext Ultra II DNA library Prep Kit for Illumina (NEB, Cat. E7645S). Two replicates were performed for each condition. Libraries were indexed with NEBNext® Multiplex Oligos for Illumina (Dual Index Primers Set 1, NEB, Cat. E7600S) and validated for quality and size distribution using a TapeStation 4200 (Agilent). Paired-end sequencing (150 bp+150 bp) was performed on Ilumina sequencer.