title: GSM1467667: oxRRBS_mESC_NT_p53_KO_3; Mus musculus; Bisulfite-Seq
library_construction_protocol: 129s-Trp53tm2Tyj/J p53+/- (Jackson Laboratory, Mt Desert Is., ME, USA) heterozygous p53+/- male and female mice were time mated. E2.5 morulas were isolated from the uteri of female mice and allowed to attach to irradiated MEF feeder layers in 2i medium. Subsequently, 2i medium was changed daily. For differentiation experiments, feeders were depleted by 30–45 minutes of incubation on 0.1% gelatin, followed by gentle aspiration of purified mESCs. For RA-induced differentiation, feeder-depleted cells were seeded on gelatin and transferred to differentiation medium containing for 16 hours followed 1μM retinoic acid (RA). RA was replenished daily for 4 days. Reduced representation bisulfite sequencing (RRBS) was performed according to a previously published protocol  (Boyle, P. et al. 2012). Briefly, genomic DNA was isolated using the PureLink genomic DNA kit (Invitrogen) according to the manufacturer’s instructions and subjected to enzymatic digestion by MSPI (New England Biolabs) for 16 hours. Klenow polymerase (New England Biolabs) was then added to each sample for end filling and poly-A tailing, followed by Truseq adapter ligation (Illumina). The library was then pooled and subjected to either bisulfite treatment as in the RRBS protocol or to an OX-RRBS protocol using the CEGX kit (Cambridge Epigentix). Validation of oxidation efficiency was done with the provided DNA sequences that were also A tailed and adapter ligated.