GSM5345600: RPE.Ctrl.S3.rep2; Homo sapiens; OTHER
Cells were washed and resuspended in 2.5 ml cold 1X PBS – 1% FBS. 7.5 ml cold 70% EtOH was added drop by drop while vortexing the samples. Fixed cells were stored at -20 degrees. Cells were pelleted at 4 degrees, resuspended in 3 ml 1X PBS – 1% FBS – 1/100 Propidium Iodide – 250 g/ml RNAse A, and incubated 1 hour RT in the dark.  After FACS sort DNA was extracted and replicating DNA immunoprecipitated with anti-BrdU. Libraries were prepared using the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences 30024) following supplier's protocol, starting with 15 ml of immunoprecipitated DNA and following the recommendation for <10 ng, using 7 cycles of PCR amplification. Library length distribution were assessed by TapeStation (Agilent) and quantified with Qubit (ThermoFisher). Libraries were single-end sequenced (75 cycles) using a NextSeq500 (Illumina).
GEO Accession
GSM5345600