GSM2610728: PrEC Repli-Seq S3 rep1; 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 FACSAriaII based on DNA content labeling using propidium iodide. DNA extraction and BrdU-labeled DNA immunoprecipitation were performed with anti-BrdU antibody (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)). ssDNA was reconstituted for the complementary DNA strand using Klenow extension with random hexamers (Random Primers DNA Labeling System, Invitrogen #18187-013) using 10 ng of ssDNA input and a 2hr incubation. Reconstituted dsDNA was rechecked for enrichment of known Early and Late loci using qPCR. Klenow-treated products were sonicated using a Sonifer 250 probe sonicator. Klenow-treated dsDNA was sent to University of Southern California Epigenome Centre Data Production Facility for 50 bp single-end sequencing on the Illumina HiSeq 2000.