XRCC1 CUT&RUN in MDA-MB-231 cells stably expressing shRFP (control) after 30 min CPT treatment, replicate 1
MDA-MB-231 cells (ATCC, HTB-26) were stably infected with pLKO.1-puro sh-RFP (control) expression vector (based on Addgene 8453). One million cells were treated with 1 µM camptothecin (CPT, Selleckchem #S1208) for 30 min to induce TOP1:DNA cleavage complex (TOP1cc) formation. Cells were harvested and subjected to CUT&RUN as described by Skene and Henikoff (protocols.io, dx.doi.org/10.17504/protocols.io.mgjc3un). After cell permeabilization, 0.5 µg of primary antibody (anti-XRCC1, Novue Biologicals, NBP1-87154) was added overnight at 4 degrees Celsius. After washing, 5 µL pAG-MNase (EpiCypher, 15-1016) was added per sample for 1 h at 4 degrees Celsius, chromatin digestion and release were performed at 0 degrees Celsius for 90 min in the presence of 2 mM CaCl2. DNA was purified using the MiniElute PCR Purification Kit (Qiagen, cat. 28004) and quantified by Qubit Flex using the dsDNA Quantification Assay Kit (Thermo Fisher, Q32851). NGS libraries were prepared using the ThruPLEX DNA-Seq Kit (Takara Bio USA, R400675), following the manufacturer's instructions. 1-10 ng DNA was used per sample and PCR cycle numbers were adjusted as recommended. Samples were bar-coded using the DNA HT Dual Index Kit – 24N (Takara Bio, R400664). Amplified libraries were purified using AMPure XP beads (Beckman Coulter, A63880) at a 1:1 (v/v) ratio. Library fragment size and concentration were measured using TapeStation (Agilent D1000 reagent, 5067-5583). Up to 18 samples were pooled at a final concentration of 4 nM per sample. Paired-end Illumina sequencing was performed on NextSeq 2000 Sequencing Systems.