GSM8683629: clinvar_screens_SpG-evoA-BE4max-R2; Homo sapiens; OTHER
The genomic DNA was extracted from these cellular samples using the following steps. Add 1 mL Lysis buffer (300 mM NaCl, 0.2% SDS, 2 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 μL RNase A (100 mg/mL) in 15 mL conical tube containing the cellular pellet followed by mixing and incubation at 65°C for 1 h. Then add 10 µL Proteinase K (10 mg/mL) to continue incubation at 55°C for 6 h or overnight. Mix with 1 mL phenol/chloroform/isoamyl alcohol solution (25:24:1) and centrifuge at 12000 rpm for 10 min before taking the upper phase. The genomic DNA was precipitated by isopropanol and washed by 75% ethanol before dissolving in nuclease-free water. These genomic DNA and plasmid library DNA were used for constructing sequencing libraries To construct plasmid library for high throughput BE evaluation, a two-step protocol was deployed. Step I: Generation of the first plasmid library containing pairs of gRNA sequences and corresponding barcodes and Step II: Generation of the second plasmid library by insertion of gRNA scaffold and target fragments.  For ClinVar SNV modeling plasmid library construction, a similar two-step protocol was deployed. The first plasmid library was generated containing designed combinations of gRNA sequences, corresponding target sequences and barcodes. The gRNA scaffold sequence was then inserted via BsmBI site by T4 DNA ligase to get the final plasmid library. The construction of drug resistance BE screen plasmid library also followed similar procedures except that the first plasmid library only contains designed gRNA sequences without corresponding target fragment.