alias: E-MTAB-6785:wt_ESC_RRBS_rep1_s
title: Illumina HiSeq 1500 sequencing; Tet1 controls maintenance DNA methylation via the regulation of Dppa3
design: Tet1 controls maintenance DNA methylation via the regulation of Dppa3
library_construction_protocol: Cells were collected by trypsinization Naïve J1 mouse ESCs were cultured and differentiated into EpiLCs as described previously (Hayashi and Saitou, 2013; Mulholland et al., 2015). In brief, for both naïve ESCs and EpiLCs defined media was used, consisting of N2B27: 50% neurobasal medium (Life Technologies), 50% DMEM/F12 (Life Technologies), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), N2 supplement (Life Technologies), B27 serum-free supplement (Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Naïve ESCs were maintained on flasks treated with 0.2% gelatin in defined media containing 2i (1 μM PD032591 and 3 μM CHIR99021 (Axon Medchem, Netherlands)), 1000 U/mL recombinant leukemia inhibitory factor (LIF, Millipore), and 0.3% BSA (Gibco) for at least three passages before commencing differentiation. For reprogramming experiments, naive media was supplemented with freshly prepared 100 µM Vitamin C  (L-ascorbic acid 2-phosphate, Sigma).  Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment.  RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol.
identifiers.namespace: Faculty of Biology Department II Ludwig Maximilians University Munich
identifiers.id: E-MTAB-6785:wt_ESC_RRBS_rep1_s
attributes.tag: Experimental Factor: cell type
attributes.value: embryonic stem cell
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype