characteristics.text: SAMN22509537
characteristics.characteristic: External Id
characteristics.text: Benjamin J. Blencowe, Donnelly Centre, University of Toronto
characteristics.characteristic: INSDC center name
characteristics.text: 2022-07-15T18:40:39.637Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-07-15T18:40:39.637Z
characteristics.characteristic: INSDC last update
characteristics.text: SRS10688177
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: SRS10688177
characteristics.characteristic: SRA accession
characteristics.text: CGR8 clonal cell lines harboring CRISPR-Cas9 mediated deletion of Puf60 exon 5 (Puf60-Ex5) 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 the upstream (Puf60_gRNA_Up1: 5′-CTTTGCAGCAGGACCGGCGA-3′) and downstream (Puf60_gRNA_Dn1: 5′-CACCTTAAAACTAACCGGCC-3′) flanking introns surrounding Puf60 exon 5 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 deletion of Puf60 exon 5, CGR8 cells were simultaneously transiently transfected with PX459-Puf60_gRNA_Up1 and PX459-Puf60_gRNA_Dn1 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 Puf60 exon 5 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 Puf60 was detected by PCR ampliﬁcation of gDNA using the following primers: Puf60 Ex5 deletion screen For: 5′- TCCACTGGGCCCTACTCTAA-3′ Puf60 Ex5 deletion screen Rev: 5′- CTTGACCCCAACCCAAGAG-3′ Positive clones were further analyzed by Sanger sequencing. Deletion of Puf60 exon 5 was confirmed by RT-PCR.
characteristics.characteristic: cloning
characteristics.text: Glutamatergic neurons at day 7 after neuronal progenitor stage
characteristics.characteristic: differentiation stage
characteristics.text: Deletion of Puf60 exon 5 (homozygous)
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: D26.DIV7.Puf60.mEx5.HOM.A
characteristics.characteristic: title