Illumina HiSeq 1500 sequencing; TETs controls maintenance DNA methylation via the regulation of Dppa3
Cells were harvested 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 the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016).  A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing.  column purification SCRB-seq adapted for bulk samples (Ziegenhain, C., Vieth, B., Parekh, S., Reinius, B., Guillaumet-Adkins, A., Smets, M., Leonhardt, H., Heyn, H., Hellmann, I., Enard, W., 2017. Comparative Analysis of Single-Cell RNA Sequencing Methods. Mol. Cell 65, 631–643.e4.)