title: Illumina HiSeq 1500 sequencing; TETs controls maintenance DNA methylation via the regulation of Dppa3
design: TETs controls maintenance DNA methylation via the regulation of Dppa3
library_construction_protocol: Cells were harvested by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h.  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.)