biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsHagit PhilipIllumina MiSeqgenotyping by high throughput sequencingHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13685A secondary custom CRISPR-Cas9 screen, encompassing the 132 genes identified in the genome-wide TP53 knockout screen along with additional 93 genes serving as positive and negative controls.biostudies-arrayexpressSequencing - MiSeq illuminaLibrary Construction - prepare mix inside a PCR hood, after cleaning the surface with DNase Away and70% EtOH. If possible, we recommend setting up at least 4 parallel PCRs for a given sample.Final contents of each reaction:• 10 μL 10x reaction buffer• 8 μL dNTP• 0.5 μL P5 primer mix, 100 μM• 1.5 μL ExTaq polymerase• up to 10 μg of genomic DNA or 200 ng of plasmid DNA• 10 μL of P7 primer 5 μM• up to 100 μL with water1. Make a master mix of reaction buffer, dNTP, P5 primer mix, taq polymerase, and water.Aliquot into a PCR plate.2. Add template DNA.3. Add a unique P7 primer to barcode each individual reaction.Thermal cycler parametersWait for block to reach 95°C before adding samples.1. 95°C, 1 minute2. 95°C 30 seconds (denaturation)3. 53°C 30 seconds (annealing)4. 72°C 30 seconds (extension)Back to step 2, total of 28 cycles5. 72°C 10 minutes6. 4°C foreverSample Collection - Wash cells with PBS x2. Trypsinize (TrypLE Select) the wells with the regular protocol. Spin down the cells after neutralizing the trypsin with a serum-containing medium. Count cellsfreeze pelletNucleic Acid Extraction - gSYNC DNA Extraction Kit (GS100)OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesHagit PhilipfalseA secondary, tailored CRISPR-Cas9 knockout library targeting the specified genes was identified through the TP53 knockout genome-wide screenA secondary custom CRISPR-Cas9 screen, encompassing the 132 genes identified in the genome-wide TP53 knockout screen along with additional 93 genes serving as positive and negative controls.2025-12-13T00:00:00Z2025-12-13T02:02:17.006Z2024-01-02T17:13:53.838ZE-MTAB-13685ERP156427E-MTAB-13682E-MTAB-13683E-MTAB-13684EFO_0002944EFO_0004170EFO_0002771EFO_0005518EFO_0004184