GSM5584943: HCT116 genomic (MCM2-mAID+ S139A MCM2) control; Homo sapiens; OTHER
DNA from cell cultures was isolated, size fractionated and subject to exunuclease digestion as described in Aladjem et al., Science 281, 1005, 1998. and Wang et al., Molecular and Cellular Biology 24:3373-86, 2004. For ChipSeq, DNA molecules were enriched using antibodies against proteins of interest. Enriched ChIPed DNA molecules were sequenced by Illumina NextSeq 75 cycle High Output kit with single ennd of 75bp. For nascent  DNA, re-replicated DNA, paired-end libraries of genomic DNA (gDNA) were prepared using Illumina TruSeq Nano DNA Library Prep kits. Control genomic DNA was fragmented to ~ 400 bp insert size on the Covaris which generates dsDNA fragments with 3' or 5' overhangs. The sheared DNA was blunt-ended and library size selection was done using sample purification beads. A single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them from ligating to each other during the adapter ligation reaction. A corresponding single 'T' nucleotide on the 3' end of the adapter provided a complementary overhang for ligating the adapter to the fragment. The indexed adapters were ligated to the ends of the DNA fragments and then PCR-amplified to enrich for fragments that have adapters on both ends. The final purified product was then quantitated by qPCR before cluster generation and sequencing.