Genome-Wide CRISPR/Cas9 Screening with vehicle 1
For PCR amplification products that do not contain any sequencing adapter sequences, after passing quality control, 100-500 ng of DNA fragments are taken to construct a high-throughput sequencing library for the Illumina platform through steps such as End Repair, 5' Phosphorylation and dA-Tailing, Adapter Ligation, Clean Up, and PCR Enrichment and Clean Up. To ensure sufficient amplification of the gRNA sequences in the sample, an adequate amount of genomic DNA should be extracted from a cell count corresponding to 300X-500X the size of the gRNA library. For each reaction, 2-3 ug of genomic DNA is used for amplification (with sufficient reaction tubes set up until the genomic DNA is fully consumed). After introducing spacers of varying lengths and partial sequencing adapters, the complete sequencing adapters and indexes are incorporated in a second round, followed by purification to obtain the high-throughput sequencing library for the Illumina platform. The constructed library is preliminarily quantified and diluted using Qubit 3.0, and the insert size and nucleic acid concentration of the library are detected using the Agilent 2100. After pooling, the effective concentration of the pooled library is accurately quantified using NanoQC.