GSM4476423: N87-DMSO-Day14-1; Homo sapiens; OTHER
The genomic DNA of gastric cancer cell lines (N87 and OE19)  was extracted ﻿using the Zymo Research Quick-gDNA MidiPrep according to the manufacturer's protocol for PCR amplification and deep sequencing of the genomic regions containing the sgRNAs was conducted. To prepare the sgRNA library for NGS, set up a PCR  reaction for each of the 10 NGS-Lib-Fwd primers and 1 NGS-Lib-KO- Rev or NGS-Lib-SAM-Rev barcode primer. After the reaction is complete, pool the PCR reactions and purify the PCR product by using the QIAquick PCR purification kit according to the manufacturer's directions.  Quantify the purified PCR product and run 2 µg of the product on a 2% (wt/vol) agarose gel. Successful reactions should yield a ~260-270bp product for the knockout library. Gel extract using the QIAquick gel extraction kit according to the manufacturer's directions. ﻿Quantify the gel-extracted samples using the Qubit dsDNA HS Assay Kit according to the manufacturer's instructions. PCR amplify the integrated sgRNA fragment from pooled gastric cacner genomic DNA to generate sgRNA library for NGS sequencing.