GSM5833997: A549_day0_rep1; Homo sapiens; OTHER
Genomic DNA was extracted from replicate samples (QIAamp Blood Midi kit) and sgRNA inserts were amplified by PCR. The sgRNAs were designed using the CRISPR-DO tool that accounted for sgRNA specificity and cutting efficiency. sgRNAs were synthesized as 73-mer oligonucleotides (CustomArray, USA), GAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC (N's denote sgRNA 20 nucleotide target sequence; sense orientation) with a total of 12,472 sequences and amplified by PCR as a pool using the following primers: TAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT GTGGAAAGGACGAAACACCG (Forward) and ACTTTTTCAAGTTGATAACG GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC (Reverse). The PCR product was purified and cloned in the lentiGuide-Puro vector using BsmBI (NEB). Ligation was performed using the NEBuilder® HiFi DNA Assembly Cloning Kit and transformed into an electrocompetent strain (Stbl4; ThermoFisher, USA) to achieve ~300x coverage. Colonies were scraped off Agar plates using LB medium. Plasmid DNA was extracted using NA0310 Sigma GenElute™ HP Plasmid Maxiprep Kit and adequate library representation of each sgRNA was confirmed by NGS.