GSM8342600: E25N_NEW_sg3_3_D14_AACYV5TM5; Homo sapiens; OTHER
Harvested cell pellets were thawed and genomic DNA were extracted by using Monarch Genomic DNA purification kit (NEB). Target region specific primer were used for PCR using Q5 High-Fidelity 2X Master Mix (NEB). Addaptor and index were added for each target region PCR product and samples were purified using AMPure XP beads and sent for sequencing. Homologous arms (HA) that are 600-1000bp upstream and downstream flanking each target region were amplied from HAP1 cells and cloned into TOPO vector; site-directed mutagenesis PCR were performed in the HA arm vector to introduce target region saturation mutations with the PAM or sgRNA recognition site -destroying to avoind re-cutting by Cas9. Pooled mutagenesis PCR product was phosphorylated and transformed into MaxEfficient DH5a competent cells, grow overnight at 37 degree and library plasmids were extracted by ZymoPUREII MaxiPrep kit.