title: GSM8756390: BJ/HRAS-ER H2B-GFP, IP S9.6, Rep 2 Day 2; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin was extracted from BJ/hTERT, BJ/hTERT/HRASG12V-ER, BJ-hTERT/HRASG12V-ER/HDAC4-FLAG, BJ-hTERT/HRASG12V-ER/RPA70-GFP and BJ-hTERT/HRASG12V-ER/H2B-GFP cells and immunoprecipitated with 3 μg of BRCA1, 6 μg of IgG, and 3μg of RNA Pol II Ser5, 3μg RPA32 or phosphoRPA32, 2 μg  γH2AX antibodies. For each ChIP, approximately 3x106 BJ cells were used. Cells were treated as indicated and DNA-protein complexes were crosslinked with 1% formaldehyde (Sigma-Aldrich) for 15 minutes at room temperature. Following quenching with PBS/glycine (125 mM) and two PBS washes, the cells were harvested, centrifuged, and then lysed on ice for 10 minutes using Swelling buffer (20 mM Tris-HCl pH 8, 85 mM KCl, 0.5% NP-40) containing freshly added PIC and PMSF. The samples were centrifuged and the resulting pellets were resuspended in Nuclei lysis buffer (10 mM Tris-HCl pH 7.5, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS) and incubated on ice for 10 minutes. Following this, the samples were sonicated at 4°C using a Bioruptor Plus (Diagenode) with 30-second pulses for 30 minutes, yielding genomic DNA fragments with an average size of approximately 300 bp. After centrifugation, the supernatants were precleared with magnetic Dynabeads protein A or G (Thermo Fisher Scientific). 1/100 of the samples were taken as input and the remainder was immunoprecipitated overnight at 4°C on a rotating system with the previously mentioned antibodies in the presence of 8 µl of magnetic Dynabeads protein G (Thermo Fisher Scientific) in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 16.7 mM Tris-HCl pH 8, 167 mM NaCl) and Nuclei lysis buffer in a 1:1 ratio. Immunoprecipitated complexes were washed three times with Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 20 mM Tris-HCl pH 8, 150 mM NaCl), twice with High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl) and once with LiCl Immune Complex Wash Buffer (250 mM LiCl, 1% NP-40, 0.2% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8) at 4°C with rotation. A final wash with TE (10 mM Tris-HCl pH 8, 1 mM EDTA) was performed. 100 µl of TE/RNase A (0.2 mg/ml, Ambion) were added to immunocomplexes and inputs, and samples were incubated for 30 minutes at 37°C. Input and beads were resuspended in a 1:1 mixture of TES (50 mM Tris-HCl pH 8, 10 mM EDTA, 2% SDS) and Reverse Crosslinking buffer (250 mM Tris-HCl pH 7.5, 62.5 mM EDTA, 1.25 M NaCl) with 0.27 mg/ml Proteinase K (ITW Reagents). Crosslinking was reversed using the following protocol in a thermocycler: 5 hours at 37°C, 10 hours at 68°C and 1 hour at 10°C. Genomic DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research), eluted in 30 µl water and chromatin was quantified using a Qubit Fluorometer. For DRIP-seq (IP with S9.6), cells were treated as indicated, and DNA-protein complexes were crosslinked with 1% formaldehyde (Sigma-Aldrich) for 10 minutes at room temperature. After quenching with PBS/glycine (125 mM) for 5 minutes at room temperature and two washes in PBS, cells were collected, centrifuged and then lysed for 10 minutes on ice with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0) supplemented with PIC and PMSF. Cells were sonicated at 4°C using Bioruptor Plus (Diagenode) with 30-second pulses for 30 minutes, resulting in an average fragment size of ~300 bp for genomic DNA. Samples were centrifuged and the supernatants were diluted with binding buffer solution (10 mM NaPO4, 140 mM NaCl, 0.05% Triton X-100) before preclearing with magnetic Dynabeads protein A or G (Thermo Fisher Scientific) for at least 1 hour at 4°C with rotation. Supernatants were collected using a magnetic bead system. 1/50 of the sample was taken as input, which was split equally for genomic DNA extraction and RNA extraction using Tri-Reagent (Sigma-Aldrich, Germany). The remaining samples were immunoprecipitated overnight at 4°C on a rotating wheel with 6 µg of S9.6 antibody, 6 µg of IgG, and 8 µl of magnetic Dynabeads protein G (Thermo Fisher Scientific). Immunoprecipitated complexes were washed three times with binding buffer and then inputs and beads were resuspended in 100 µl TES (50 mM Tris-HCl pH 8, 10 mM EDTA, 2% SDS) and Reverse Crosslinking salt mixture (250 mM Tris-HCl pH 7.5, 62.5 mM EDTA, 1.25 M NaCl, 0.27 mg/ml Proteinase K (ITW Reagents)). After incubation in a thermocycler (5 hours at 37°C, 10 hours at 68°C, 1 hour at 10°C), nucleic acids were purified with ChIP DNA Clean & Concentrator kit (Zymo Research), eluted in 30 µl water and quantified using a Qubit Fluorometer. dsRNA were removed prior to library preparation by treating samples with 10u RNAseIII (Ambion). DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing. To generate ChIP-seq libraries with the DNA SMART ChIP-Seq Kit, a priming site was added to the 3' end of the DNA template using the Terminal Deoxynucleotidyl Transferase. This was followed by annealing of a Poly(dA) Primer, which anneals to the T-tail. This primer was then used by the SMARTScribe RT to copy the DNA strand. Sequencing libraries were then generated by PCR-mediated addition of Illumina adapters. DNA nanoball (DNB) technology was applied for library preparation of samples subjected to BGI-seq 500 sequencing following manifacturer (Roche)