Repli-Seq of the HeLa-JVM cell line, early fraction Replicate 1
HeLa-JVM cells were pulse-labelled with BrdU, fixed, stained with propidium iodide, and separated into early S-phase and late S-phase fractions. Approximately 200,000 cells were collected for each fraction. Cells were lysed and DNA was extracted with a Monarch Genomic DNA Purification Kit. DNA was sheared with a Covaris M220 ultrasonicator to produce an average fragment length of 200bp. Fragmented DNA was ligated to the adaptors from a NEBNext Ultra DNA Library Prep Kit for Illumina. BrdU immunoprecipitation was performed on the adaptor-ligated DNA. The DNA-antibody complex was precipitated and the DNA released by proteinase K digestion. DNA was purified and then indexed and amplified with a NEBNext Multiplex Oligos for Illumina kit. The amplified indexed DNA was purified with AMPure XP beads. Libraries were sequenced on an Illumina NovaSeq 6000 platform.