TOP1 CAD-Seq in MDA-MB-231 cells after 5 min CPT treatment
Ten million MDA-MB-231 cells (ATCC, HTB-26) were treated with 20 µM camptothecin (CPT, Selleckchem S1208) for 5 min to stabilize existing TOP1:DNA cleavage complexes (TOP1cc). Cells were pretreated with 10 μM MG132 (Sigma, 474790) for 30 min to prevent repair-associated TOP1cc degradation. Cells were harvested and sonicated using a Fisher Scientific Sonic Dismembrator Model 100 at level 2 in three 5 seconds on, 5 seconds off cycles with 30 second intervals. DNA was ethanol-precipitated following the TOP1 Covalent Adduct Detection (CAD)-Seq procedure as described by Kuzin et al (PMID: 35942340). DNA was fragmented using the Covaris ME220 High Cell protocol. TOP1cc DNA fragments were immunoprecipitated at 4 degrees Celsius overnight using 2 µg of anti-TOP1 antibody (Abcam, ab109374) conjugated to protein A/G beads (Thermo Fisher, 88803). After washing and proteinase K digest, DNA was purified using the MiniElute PCR purification kit (QIAGEN, 28006) and quantified by Qubit Flex using the Qubit™ 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. Single- or paired-end Illumina sequencing was performed on NextSeq 2000 Sequencing Systems.