title: GSM9054689: MDAMB231 breast cancer cells, NT, rep2; Homo sapiens; OTHER
library_construction_protocol: sBLISS was conducted as previously described (M Bouwman, B. A. et al. Genome-wide detection of DNA double-strand breaks by in-suspension BLISS. Nat Protoc doi:10.1038/s41596-020-0397-2., Hidmi, O., Oster, S., Shatleh, D., Monin, J. & Aqeilan, R. I. Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing. STAR Protoc 5, (2024).). In summary, 106 cells were fixed in 2% paraformaldehyde in 10% FCS/PBS for 10 minutes at room temperature. The fixation was quenched with 125 mM glycine for 5 minutes at room temperature, followed by another 5 minutes on ice and two washes in ice-cold PBS. Cells were lysed for 60 minutes on ice, and their nuclei were permeabilized for 60 minutes at 37°C. Next, nuclei were rinsed twice with CutSmart Buffer containing 0.1% Triton X-100 (CS/TX100), and double-strand break (DSB) ends were blunted in situ using NEB's Quick Blunting Kit for 60 minutes at room temperature. The blunted nuclei were then washed twice with 1x CS/TX100 before in situ ligation of the sBLISS adapters to the DSB ends. Adaptor ligation was carried out with T4 DNA Ligase for 20-24 hours at 16°C, with BSA and ATP added. Following ligation, the nuclei underwent two washes with 1x CS/TX100, and genomic DNA was extracted using Proteinase K at 55°C for 14-18 hours. Proteinase K was then heat-inactivated for 10 minutes at 95°C, followed by extraction using Phenol:Chloroform:Isoamyl Alcohol, Chloroform, and ethanol precipitation. Preparation of sBLISS libraries: The purified DNA was sonicated in 100 μL of ultra-pure water using Covaris M220 for 60 seconds. Sonicated samples were concentrated with AMPure XP beads (Beckman Coulter), and fragment sizes were evaluated using a BioAnalyzer 2100 (Agilent Technologies), targeting a range of 300 bp to 800 bp with a peak around 400-600 bp. The sonicated DNA was then in vitro transcribed using the MEGAscript T7 Kit for 14 hours at 37°C. After RNA purification and ligation of the 3'-Illumina adaptors, the RNA underwent reverse transcription. The final library indexing and amplification step was performed with NEBNext® Ultra™ II Q5® Master Mix.