title: GSM8878509: Synchronous S1, LIG1/LIG3-mAID, 5-Ph-IAA; Homo sapiens; OTHER
library_construction_protocol: Approximately 7 million cells were collected for each sample and processed to purify genomic DNA using Blood & Cell Culture DNA Midi Kit (QIAGEN, #13343) with 100/G Genomic-tips. To examine single-stranded Okazaki fragments, 30 µg of genomic DNA was mixed with 2×RNA loading dye (95% formamide, 0.02% SDS, 0.04% Orange G, 1 mM EDTA) and denatured at 70°C for 10 min followed by 10 min incubation on ice. The DNA samples were loaded onto a 1% alkaline agarose gel and electrophoresed for 45 min at 70 V at 4°C. Gels were neutralised in 1×TAE buffer for 30 min and then stained with SYBR Gold (Invitrogen, #S11494) for 1 h at room temperature. Single-stranded DNA ranging from 100 to 650 bp was excised from the gel, which was subsequently processed for ssDNA purification using NucleoSpin Gel and PCR Clean-up Kit (Takara, #740609) with NTC buffer (Takara, #U0654A). Quantification of ssDNA was performed using Quant-iT OliGreen ssDNA Assay Kit (Invitrogen, #O7582). To convert purified ssDNA to dsDNA, 50-100 ng of ssDNA in 30 µl was mixed with 5 µl of 10×NEBuffer 2 (NEB, #B7002S) and 5 µl of 3 mg/ml 8N random primers (Eurofins). The mixture was incubated at 95°C for 5 min followed by cooling for 5 min on ice, and then supplemented with 5 µl of 2 mM dNTP mix (without dTTP, Roche, #11969064001) plus dUTP (Roche, #11934554001), 2.5 µl of 5,000 U/ml Klenow DNA polymerase (NEB, #M0210S), and nuclease-free H2O to a final volume of 50 µl. The supplemented mixture was incubated at 37°C for 20 min. Reactions were stopped by adding 5 µl of 0.5 M EDTA. The dsDNA was purified using AMPure XP beads (Beckman Coulter, #A63881) and quantified with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, #P7589) before proceeding to library preparation. Sequencing libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, #E7645S) according to the manufacturer's instructions. Sequencing was performed using the Illumina NovaSeq 6000 platform with 100 bp paired-end reads for approximately 100 million read pairs per sample.
identifiers.namespace: GEO
identifiers.id: GSM8878509_r1