characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2020-06-29T04:03:40Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2019-10-21T16:29:52Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA6104681
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: 2019-10-21T16:29:52Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS3905361
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6797:T12CM_EpilC_rep3
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: epiblast like cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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.)
characteristics.characteristic: description
characteristics.text: blastocyst
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000358
characteristics.characteristic: developmental stage
characteristics.text: Tet1Tet2CM
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: T12CM_EpilC_rep3
characteristics.characteristic: title