GSM4784046: HCT116 DKO1 Repli-Seq R2 G2; Homo sapiens; OTHER
Cells were labeled with BrdU (50uM, Sigma, #B5002) for two hours. Labeled cells were sorted into 6 fractions across the cell cycle (G1b, S1, S2, S3, S4, G2M) on the FACSAria III based on DNA content labeling using propidium iodide. DNA extraction and BrdU-labeled DNA immunoprecipitation were performed with anti-BrdU antibody (40uL of 25ug/mL, BD Pharmingen, #555627). Validation of BrdU immunoprecipitation was carried out using qPCR on known Early (BMP1) and Late (DPPA2) loci (primers from Ryba et. al. (2011)). 10 ng of ssDNA was used as input for the Epicenter EpiGnome™ Methyl-Seq Kit (Illumina, EGMK81312, later called the TruSeq® DNA Methylation kit) and processed according to manufacturer's instructions. The ssDNA was not bisulphite converted prior to library preparation. Libraries were sequenced on the HiSeq 2500 as 50bp single-end reads. Repli-Seq