GSM5502572: TOP1cc CAD-seq MYC-mAID auxinole rep 1; Homo sapiens; ChIP-Seq
Cells in suspension were collected by centrifugation and adherent cells were scraped in M buffer (9.3mM 870 Tris-HCl pH6.5, 18.6 mM EDTA, 5.59M guanidine thiocyanate, 0.93% DTT, 0.93% Sarcosyl, 3.72% Triton) to a final concentration of 5x106cells/ml and briefly sonicated with Bandelin probe sonicator at 20% amp for 3 cycles 30sec ON, 30 sec OFF. 2.5 ml DNA and bound proteins were precipitated with 1.25 ml EtOH at -20°C, washed thrice in wash buffer (20mM Tris-HCl pH7.5, 50mM NaCl, 1mM EDTA, 50% EtOH). The resulting pellet was dried for 5 minutes and resuspended in TE-SDS (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 0.1% SDS) and sonicated with Covaris ME220 sonicator for 5 minutes at High Cell protocol in milliTUBE–1 mL with AFA Fiber. For immunoprecipitation 1.5 μg anti-TOP1 (ab109374) or 3 μg of 1:1 mixture of anti-TOP2A (sc166934 and ab52934) antibodies were mixed with 30 μl Protein A/G magnetic beads (Pierce, 88803) and incubated at 40C for 6 hrs with controlled rotation. DNA and bound complexes from 1×107 cells were added to the Protein A/G-antibody complexes and incubated overnight at 40C with rotation. Washing was performed as described above but only once 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 60ºC. Samples were then purified by PCR purification Kit (Qiagen). DNA from CAD-seq samples was quantified with the Qubit dsDNA HS Assay Kit. To cleave off covalently bound tyrosyl residues from TOP2cc CAD-seq DNA samples, the samples were additionally treated with ExoVII (0.5U per 10 ng of DNA) and purified by AMPure XP beads. 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.