GSM7469351: HDR Pool 3 GL2 - Rep 3 gfp +; Homo sapiens; OTHER
Inverse PCR reactions of the plasmid encoding the BRCA1 cDNA generate full length product, but one of the codons in the PCR oligonucleotide contains NNK (where N = A, C, G, T and K = G, T) in place of the terminal codon encoded by the oligonucleotide. For the 97 codons analyzed in this study, 97 different inverted oligonucleotide pairs were separately prepared and subjected to PCR. The PCR reactions were then pooled and circularized by the action of DNA ligase. An oligonucleotide containing a 16 bp degenerate oligonucleotide barcode was inserted upstream of the BRCA1 coding sequence, and long-read sequencing using a PacBio Sequel paired each barcode with a different BRCA1 variant. The plasmid library for pools 3 was transfected along with the FLP-In recombinase into a HeLa derived cell line that contained in its genome a single FLP-In Recombinase Target (FRT) sequence  and a single DNA sequence for measuring homology directed repair (HDR) of DNA double-strand breaks. The HDR assay is initiated by transfecting a plasmid encoding the I-SceI endonuclease, which generates a DNA cut in one of the defective GFP sequences, and if the cell is competent for HDR, recombination repairs the defective GFP coding sequence, and the cell becomes GFP-positive. We performed this HDR assay under two conditions. In the first condition, we have transfected a control siRNA, and the cells contain endogenously expressed wild-type BRCA1. In the second condition, we have transfected an siRNA targeting the 3'-UTR of the endogenously expressed BRCA1 mRNA, and the cell is then dependent on the BRCA1 variant expressed from the FRT site. GFP-positive cells were separated from GFP-negative cells using flow sorting, and the barcode in the genome of the cells was isolated by PCR and analyzed by short read sequencing. The frequency of the barcode in the GFP-positive cells (functional for HDR) is compared under the conditions of depletion of the endogenous BRCA1 protein using the siRNA targeting the BRCA1 3'-UTR to the condition of the control siRNA. From the abundances of the barcodes in the various samples, we calculated a functional score using the program Enrich2.