characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-08-01T17:04:24Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T22:13:32Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA3146539
characteristics.characteristic: External Id
characteristics.text: London Research Institute CRUK
characteristics.characteristic: INSDC center name
characteristics.text: 2015-08-01T17:04:24Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T22:13:32Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS617402
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-3125:WT_nuclear_RNA_seq_rep1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: J1 (129S4/SvJae) and 129SvJae/Cast WT mESCs were grown under standard ESC culture conditions. Ring1A−/−;Ring1Bfl/fl;Rosa26::CreERT2 mESCs (Ring1A-KO) were cultured as described previously in Endoh et al. Dishes were coated with 0.1 % gelatine and irradiated mouse embryonic fibroblasts (MEFs). For harvesting, ESCs were trypsinized and pre-plated twice for 30 min to remove contaminating feeder cells. Mouse ESCs were washed in PBS and approximately 30-50 x 106 ES cells were lysed for 5 min in 0.5 ml cold buffer RLN (50 mM TrisHCl pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.4% Igepal). Nuclei were pelleted by spinning at 300g for 10 min at 4°C. Nuclear RNA was isolated using TRIsure (Bioline), treated with DNaseI (Roche) and re-purified using an RNeasy Mini Kit (Qiagen). Strand-specific RNA-seq libraries were prepared as described previously by marking the second strand with dUTP but with some modifications8,9. 250 ng nuclear RNA was fragmented using a Covaris E220 instrument at standard RNA settings for 60 sec. Fragmented RNA was precipitated and first strand synthesis was carried out using SuperScript III (Invitrogen) with 4 µg of actinomycin D (Sigma). Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas). Following purification on QIAquick columns (Qiagen), TruSeq Illumina adapters were ligated with T4 DNA Ligase, (Enzymatics). Libraries were purified on QIAquick columns, treated with USER (NEB) to destroy the second strand and size selected using AMPure XP beads. Libraries were amplified with 9-11 PCR cycles.
characteristics.characteristic: description
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
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: wild type
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: phenotype
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: J1
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0004990
characteristics.characteristic: strain
characteristics.text: WT_nuclear_RNA_seq_rep1
characteristics.characteristic: title