pre-dose, 25 nM camptothecin (CPT), 1 h, harvest 24 h later
Sequenced DNA Library
library_name
GSM6533398
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Top1 ChIP was performed on RPE-1 cells as described previously (Cameron et al. 2021) with minor modifications. Briefly, after treating with the challenging dose or 24 h after treatment with pre-dose, cells were treated for 5 minutes with 1 μM CPT to increase the probability to trap Top1 on the DNA. Cells were crosslinked with 1% formaldehyde (Thermo Fisher, 28906) for 5 min. Cross-linking was stopped by the addition of 125 mM glycine (Sigma, 50046) and cells were washed twice with cold PBS. After harvesting cells, the pellet was washed once with PBS plus 0.5% BSA and resuspended in TE-SDS 0.1% (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, SDS 0.1%) with complete protease inhibitor tablet (Roche) to a final concentration of 1x10000000 cells/ml. Samples were sonicated with a Bandelin probe sonicator (30% amplitude, 4 times 30'' ON/ 30'' OFF) and then with Covaris ME220 sonicator for 10 min using the 1 ml High Cell protocol (Peak power: 75, Duty % factor: 15, Cycles/Burst: 1000, Average power: 11.25) to produce chromatin fragments of 200-500 bp on average. After centrifugation, sonicated extracts were adjusted to the conditions of RIPA buffer (10mM Tris pH 8.0, 1mM EDTA pH 8.0, 1% Triton X100, 0.1% SDS, 200 mM NaCl, Na Deoxycholate 0.1%). 2 μg of anti-Top1 (ab109374) were mixed with 30 μl of Protein A/G magnetic beads (Pierce, 88803) and incubated at 4°C for 6 h with rotation. Chromatin from 10×1000000 cells was added to the Protein A/G-antibody complexes and incubated overnight at 4°C with rotation. Immunoprecipitates were washed twice with RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% TritonX100, 0.1% Na-Deoxycholate, 0.1% SDS, 200 mM NaCl); twice with RIPA buffer plus 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. The beads were then resuspended in 125 μl TE plus 0.5% SDS supplemented with proteinase K (500 μg/ml, Thermo Fisher 25530049) and incubated overnight at 65ºC. The DNA was recovered from the eluate using the QiaQuick PCR purification kit and finally dissolved in Tris-HCl ph 8.5. All ChIP-seq experiments were performed in duplicates. 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, R400676). Size selection was performed in the range of 200 – 700 bp with AMPure XP beads (Beckman, A63880) and confirmed using the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626) on the Agilent 2100 Bioanalyzer. Libraries were pooled and sequenced using the NextSeq 500/550 High Output Kit v2.5 (Illumina, 20024906). The sequencing run was Single End and Dual Index with 75 bp reads.