GSM5502560: MYC ChIP-seq TOP2A-mAID auxinole rep 1; Homo sapiens; ChIP-Seq
For RNAPII and MYC, fter harvesting cells by scraping, the pellet was washed once with PBS plus 0.5% BSA and resuspended in RIPA buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS, 200mM NaCl, with the addition of protease inhibitor cocktail) to a final concentration of 1x107 cells/ml. For TOP2A cells were directly scapred in TE-SDS 0.25%. Samples were sonicated with a Bandelin probe sonicator to produce chromatin fragments of 400 bps on average. For normalization of RNAPII and MYC ChIP-seq 20 ng spike-in chromatin (Active Motif, 53083) was added per 1x107 cells. After centrifugation, extracts were immunoprecipitated wtih 2 μg of anti-MYC antibody (ab32072) or 2 μg of anti-RNAPII (ab5408) antibody and 1 μg of spike-in antibody (Active Motif, 61686) or 2 μg of anti-FLAG (F1804) antibody were mixed with 35 μl Protein A/G magnetic beads (Pierce, 88803) and incubated at 40C for 6 hrs with controlled rotation. Chromatin from 1x107 cells was added to the Protein A/G-antibody complexes and incubated with rotation overnight at 40C. Samples were washed twice with RIPA buffer, twice with RIPA buffer containing 300 mM NaCl, twice with LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-Deoxycholate) and twice with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). The beads were then resuspended in 125 μl TE plus 0.25% SDS supplemented with proteinase K (500 μg/ml, Roche) and incubated overnight at 65ºC. DNA was recovered from the elute by phenol:chloroform (25:24:1) (Sigma) extraction followed by ethanol (100%) precipitation in the presence of 20 μg of GlycoBlue (Invitrogen) and dissolved in TE. To cleave off covalently bound tyrosyl residues from TOP2A ChIP samples, the samples were additionally treated with ExoVII (1U per 10 ng of DNA) and purified by PCR purification Kit (Qiagen). DNA from ChIP was quantified with the Qubit dsDNA HS Assay Kit. Sequencing libraries were created according to the ThruPLEX DNA-seq kit protocol (Takara). Size selection was performed in the range of 200-700bp with AMPure XP beads and confirmed using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer. Libraries were pooled and sequenced using the NextSeq 500/550 High Output Kit v2.5 (Illumina). The sequencing run was Single End and Dual Index with 75 bp reads.