characteristics.text: 2025-11-20
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: South China University of Technology
characteristics.characteristic: INSDC center name
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS27173421
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-15874:293T-GeCKOv2-Amplicon-sg3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: HEK293T
characteristics.characteristic: cell line
characteristics.text: embryonic kidney epithelial-like cells
characteristics.characteristic: cell type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: The human GeCKOv2 lentiviral library (GENEWIZ, Suzhou), containing 122,756 sgRNAs targeting 19,050 genes (6 sgRNAs per gene), 1,864 miRNAs (4 sgRNAs per miRNA), and 1,000 NonTarget controls, was transduced into 3 × 10⁸ HEK293T cells at an MOI of 0.3. After 24 hours, cells were selected with 1 μg/mL puromycin. After selection, surviving cells were expanded for 4 days, the resulting IPGRM cell pool was subjected to secondary editing. In brief, a total of 6.3×107 IPGRM library cells were seeded into three T175 flasks 24 hours before transfection and cultured to 70-80% confluence. Each flask was co-transfected with 7.5 μg pCMV-Cas9 and 15 μg pU6-sgRNA plasmids targeting either sg3 or sg81 Genomic DNA was extracted using MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa).  PCR amplification were performed in 20 μL reactions containing 40 μg genomic DNA, 0.4 μM of each primer, and Q5 Hot Start HiFi PCR Master Mix (NEB). Cycling conditions were: 98 °C for 2 min; 20 cycles of 98 °C for 10 s and 65 °C for 45 s; and a final 5 min extension at 65 °C. PCR products were purified using Cycle Pure Kit (Omega) and size-selected by Gel Extraction Kit (Omega).
characteristics.characteristic: description
characteristics.text: embryonic stage
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: Pooled CRISPR/Cas9 knockout (GeCKOv2 library); secondary locus-specific editing with sg3
characteristics.characteristic: genetic modification
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: kidney
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: 293T-GeCKOv2-Amplicon-sg3
characteristics.characteristic: title