title: GSM9282432: TGTGCCCA_F5; Homo sapiens; OTHER
library_construction_protocol: Primary human peripheral blood NK cells from two healthy donors were first transduced with HER2-CAR lentivirus and then with the ORF library lentivirus at an MOI of ~0.2. A total of 20 million library-transduced CAR-NK cells were injected into each HT29 tumor-bearing mouse (n = 5 mice for Donor 0957 and n = 3 for Donor 0958 for each time point). Genomic DNA was extracted from tumors at day 3 and 6 post-treatment, as well as from pre-injection cells, and subjected to NGS to quantify ORF and UMI barcode representations. To generate the ORF library, the top 35 gene targets (cDNA length ≤ 1 kb) were chosen from the CRISPRa screen, along with five fluorescent reporter genes as null controls. The ORF library was synthesized as individual eBlocks (IDT), pooled at equimolar ratios, and amplified with primers containing four-nucleotide random barcodes (UMIs; NNNN) to introduce molecular redundancy targeting the same gene. The ORF expression vector plasmid was bought from Addgene (#145026). The barcoded ORF library was pool-cloned into the expression vector using Gibson assembly.
identifiers.namespace: GEO
identifiers.id: GSM9282432_r1