biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsYanbin WanIllumina NovaSeq 6000genotyping by high throughput sequencingHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15874Cells were transduced with a genome-wide CRISPR knockout library to introduce gene disruptions across the genome. After Cas9-induced DNA double-strand breaks, repair outcomes were captured and quantified using high-throughput sequencing. This experiment aims to identify genetic factors influencing specific DNA repair outcome patterns such as 1-bp insertions, microhomology-mediated deletions, and large insertions.biostudies-arrayexpressNucleic Acid Extraction - Genomic DNA was extracted using MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa).Library Construction - 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).Sequencing - Purified amplicons were submitted to GENEWIZ for paired-end 150 bp (PE150) sequencing, generating 46 Gb of data per sample.Sample Collection - 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 sg81OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesYanbin WanfalseGenome-wide CRISPR knockout library screening coupled with high-throughput sequencing to analyze DNA repair outcomesCells were transduced with a genome-wide CRISPR knockout library to introduce gene disruptions across the genome. After Cas9-induced DNA double-strand breaks, repair outcomes were captured and quantified using high-throughput sequencing. This experiment aims to identify genetic factors influencing specific DNA repair outcome patterns such as 1-bp insertions, microhomology-mediated deletions, and large insertions.2025-11-20T00:00:00Z2025-11-20T02:02:22.124Z2025-11-01T17:33:17.085ZE-MTAB-15874ERP183501EFO_0002944EFO_0004170EFO_0002771EFO_0005518EFO_0004184