GSM5502580: TOP1cc cross-CAD-seq untr ETO MG132 rep 2; Homo sapiens; ChIP-Seq
Cells were lysed with 10 ml of STE buffer (2% SDS, 0.5 M Tris-HCl pH 8.1, 10 mM EDTA) and were disrupted using a Dounce homogenizer tight pestle shearing apparatus. Samples were extracted with Phenol: chloroform and was precipitated by adding 2.5 volume ethanol 100% and 1/10 volume sodium acetate (3M, pH 5.2). Pellet was washed by ethanol 70%, dried and finally dissolved in elution buffer (Tris pH 8.5). samples were sonicated with a Bandelin probe sonicator to produce chromatin fragments <1 kb on average, were further purified through QIAquick PCR purification kit (QIAGEN) using columns (provided in the kit) such that the total DNA bound per column was not exceeded 10 mg (each sample were divided into 3 columns) and was eluted in elution buffer (Tris-HCl pH 8.5). 2 µg of anti-TOP1 antibody (ab109374) were mixed with 40 ml Protein A/G magnetic beads (Pierce, 88803) and incubated at 40C for 6 hrs with controlled rotation. Preeluted CAD samples from 1×107 cells were added to the Protein A/G-antibody complexes and incubated with rotation overnight at 40C. Washing was performed as described for the ChIP protocol but twice with every buffer and always in presence of 0.1% SDS. The beads were then resuspended in 100 μl TE plus 0.25% SDS supplemented with proteinase K (500 μg/ml, Roche) and incubated for 4 hrs at 650C. Samples were then either purified by PCR purification Kit (QIAGEN) or extracted with Phenol: Chloroform and precipitated with EtOH (1/10 volume of NaoAc 3M, 2.5xVolume 100% EtOH, 1.5μl Glycogen). DNA from the Cross-CAD CHIP samples quantified with the Qubit dsDNA HS Assay Kit. To cleave off covalently bound tyrosyl residues from cross-CAD samples, the samples were additionally treated with ExoVII (0.5-1U per 10 ng of DNA) and purified by PCR purification Kit (QIAGEN). Cross-CAD libraries were prepared with Swift Bioscience AccelNGS 2S Plus DNA kit, pooled together and sequenced on NovaSeq in Single End mode with 101 bp reads.