GSM7844095: Rex1-EmGFPd2 Rfc2 gRNA#2 mESCs, BrdU-IP Early; Mus musculus; OTHER
We followed the BrdU-IP-based protocol as described (Ryba et al., 2011). 75% ethanol fixed cells were stained with Propidium iodide (Nacalai tesque, 29037), then early-S phase and late-S phase cell population (at least 10,000 cells/fraction) were sorted by a Sony SH800 cell sorter (ultra-purity mode). For nocodazole/5-Ph-IAA removal experiments, only early-S phase cells were collected. Extracted DNA was sonicated by a Bioruptor BR2012A (Sonic Bio). BrdU-incorporated DNA was immunoprecipitated using anti-BrdU antibody (BD Biosciences Pharmingen, #555627). BrdU-immunoprecipitated DNA samples were subject to whole-genome amplification (WGA) with a SeqPlex kit (Sigma, SEQXE). NGS libraries were constructed from early- and late-replicating DNA after WGA with an NGS LTP Library Preparation Kit (KAPA, KK8232) according to the manufacturer's instructions