characteristics.text: SAMN22509529
characteristics.characteristic: External Id
characteristics.text: Benjamin J. Blencowe, Donnelly Centre, University of Toronto
characteristics.characteristic: INSDC center name
characteristics.text: 2022-07-15T18:40:39.500Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-07-15T18:40:39.500Z
characteristics.characteristic: INSDC last update
characteristics.text: SRS10688185
characteristics.characteristic: INSDC secondary accession
characteristics.text: live
characteristics.characteristic: INSDC status
characteristics.text: Generic
characteristics.characteristic: NCBI submission model
characteristics.text: Generic.1.0
characteristics.characteristic: NCBI submission package
characteristics.text: SRS10688185
characteristics.characteristic: SRA accession
characteristics.text: CGR8 clonal cell lines harboring a CRISPR-Cas9 mediated ~1300bp deletion within the Rbm38 locus (Rbm38 KO) were generated essentially as described previously (Gueroussov et al., 2015; Ran et al., 2013). Brieﬂy, 20nt guide sequences were designed to have minimum off-target effects using the online CRISPR design tool provided by the Zhang laboratory (http://tools.genome-engineering.org). Guide sequences targeting within Rbm38 (Rbm38_gRNA2: 5′-GCTCCCCGAGTGTGTTTCCG-3′) and (Rbm38_gRNA4: 5′-CCTTGATCCAGCGCACCTAC-3′) were selected. The guide sequences were cloned individually into the pSpCas9(BB)-2A-Puro (PX459) vector (Addgene #48139), as described previously (Ran et al., 2013). For the generation of Rbm38 knockout cells, CGR8 cells were simultaneously transiently transfected with PX459-Rbm38_gRNA2 and PX459-Rbm38_gRNA4 plasmids, using Lipofectamine 2000 (Thermo Fisher Scientiﬁc) following the manufacturer’s instructions. 24 hours post-transfection, 1 μg/ml puromycin was applied to the media to select for transfected cells. Five days post-transfection the cells were plated on gelatin-coated 96-well plates at a concentration of 0.6 cells/well in order to achieve clonal mESC lines. Ten days later, the clones were expanded and screened for editing of the Rbm38 locus. For screening cell clones, gDNA was extracted using the Phusion Human Specimen Direct PCR Kit (Thermo Fisher Scientiﬁc) as per the manufacturer’s recommendations. Editing of Rbm38 was detected by PCR ampliﬁcation of gDNA using the following primers: Rbm38 KO screen For: 5′-GGACAGCGTGGGGAACTC-3′ Rbm38 KO screen Rev: 5′-CCACACCCCCAATACACAAT-3′ Rbm38 KO screen Internal: 5′- GCTGTCCAGGCCATCAGTA-3′ Positive clones were further analyzed by Sanger sequencing. Rbm38 protein depletion was confirmed by Western Blot.
characteristics.characteristic: cloning
characteristics.text: Glutamatergic neurons at day 7 after neuronal progenitor stage
characteristics.characteristic: differentiation stage
characteristics.text: Deletion of Rbm38
characteristics.characteristic: mutation
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: Glutamatergic neurons derived in vitro from mouse embryonic stem cells
characteristics.characteristic: source_name
characteristics.text: Glutamatergic neurons derived in vitro from mouse embryonic stem cells
characteristics.characteristic: tissue
characteristics.text: D35.DIV7.Rbm38KO.B
characteristics.characteristic: title