GSM7065897: KO U2OS nascent DNA sequencing replicate 3; Homo sapiens; OTHER
Genomic DNA was prepared using DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer's instructions. Purified DNA was sonicated to obtain fragments of average size of 200-300 bp. DNA was then concentrated using SPRIselect beads (Beckman Coulter) and eluted in TE buffer. 1 mg of DNA was used with NEBNext Ultra DNA Library Prep Kit (New England Biolabs) and NEBNext Multiplex Oligos for Illumina kit for adaptor ligation, as instructed by the manufacturer's protocol. DNA was then purified using SPRIselect beads, and eluted in water. BrdU labelled E. coli genomic DNA was processed in the same way for library preparation, and used as a spike control for BrdU immunoprecipitation. DNA was denatured at 95°C for 5 min and quickly cooled on ice. It was then incubated anti-BrdU antibody (BD347580) for 2 h in IP buffer (PBS, 0.0625% Triton X-100), after which Protein G Dynabeads were added for further 2 h incubation. Beads were pelleted and washed in IP and TE buffers using a magnetic rack, after which immunoprecipitated DNA was eluted by incubating beads in elution buffer (TE, 1% SDS) for 15 min at 65 °C. DNA was then purified using SPRIselect beads, and amplified for 15 cycles with Illumina indexing primers using NEBNext Ultra II kit and NEBNext Multiplex Oligos for Illumina kit. Amplified libraries were cleaned using SPRIselect beads and used for sequencing on NovaSeq 6000 platform.