sgRNA sequencing of Non-kinase Orchestrated VEGFR2–ERK Hyperactivation as a Dependency and Pharmacological Target in Tumor Angiogenesis
To identify genes driver resistance of apatinib on human umbilical vein endothelial cells (HUVECs). The CRISPR-Pool SAM human library, containing 70290 sgRNAs targeting 23430 genes were constructed in MS2-P65-HSF1. After the cells were treated with apatinib for another week and their genomic DNA was extracted. The sgRNA fragments were amplified by PCR. After passing quality control, 20–50 ng of DNA fragments are used 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.