characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2020-06-29T04:03:40Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2019-10-21T16:29:53Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA6104702
characteristics.characteristic: External Id
characteristics.text: Faculty of Biology Department II Ludwig Maximilians University Munich
characteristics.characteristic: INSDC center name
characteristics.text: 2020-06-29T04:03:40Z
characteristics.characteristic: INSDC first public
characteristics.text: 2019-10-21T16:29:53Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS3905382
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6797:T2CM_ESC_rep4
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: J1
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0004990
characteristics.characteristic: cell line
characteristics.text: naive embryonic stem cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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.)
characteristics.characteristic: description
characteristics.text: blastocyst
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000358
characteristics.characteristic: developmental stage
characteristics.text: Tet2CM
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: inner cell mass
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000087
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: 129S4/SvJae
characteristics.characteristic: strain
characteristics.text: T2CM_ESC_rep4
characteristics.characteristic: title