characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2021-10-02T00:19:07Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2021-10-02T00:19:07Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA10159229
characteristics.characteristic: External Id
characteristics.text: Human embryo and stem cell laboratory, The Francis Crick Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2021-10-02T00:19:07Z
characteristics.characteristic: INSDC first public
characteristics.text: 2021-10-02T00:19:07Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS7815714
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-10958:UI_Day2_3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: H9
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003045
characteristics.characteristic: cell line
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: RNA was isolated using TRI reagent (Sigma) by applying 1 ml reagent in-plate to one 6-well of cells and scraping to collect all material. Doxycycline-inducible overexpression of HA-tagged proteins was achieved using the Lenti-X Tet-On 3G Inducible Expression System (Clontech) following the manufacturer’s protocol, and as outlined previously (Wamaitha et al., 2015). Lentiviral packaging was achieved using 7 μg of transgene-containing plasmid and the Lenti-X Packaging Single Shot reagents. Lentiviral supernatant was harvested after 48hrs and concentrated by ultracentrifugation. To produce stably transduced cells, hESCs were plated under standard conditions and changed into fresh medium the following morning. 24hrs post-plating, 10 μl concentrated virus was added to hESCs for transduction overnight (~16hrs). hESCs were dual selected with 150 μg/ml G418 and 0.5 μg/ml puromycin 48hrs post-transduction. For the RNA-seq experiments, KLF17-inducible hESCs were plated as normal and induction initiated after 24 hours by addition of 1 μg/ml Dox to the culture medium (mTeSR1). At ~30 hours, a day 0 (pre-induction) control sample was collected, then both induced (+Dox) and uninduced (UI) samples were collected at 24hr intervals from 48hrs (day 1 post-induction) until 144hrs (day 5 post-induction). TRI reagent samples were subjected to phenol/chloroform-based extraction, and DNase I-treated (Ambion). cDNA was synthesised using a Maxima first strand cDNA synthesis kit (Fermentas). DNase-treated RNA was supplied to the in-house sequencing facility and subjected to library construction via the KAPA mRNA (polyA) HyperPrep protocol.
characteristics.characteristic: description
characteristics.text: embryo stage
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: Dox-inducible KLF17_HA transgene
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: blastocyst
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000358
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: UI_Day2_3
characteristics.characteristic: title