title: GSM5106491: High2; Homo sapiens; OTHER
library_construction_protocol: The genomic DNA of every replicate from 4 X 106 live cells was isolated using QIAamp DNA mini kit (Qiagen). The genomic DNA was subjected to 2 step PCR. For the first PCR, the amount of input gDNA was more than 200X coverage of designed gRNA library (assuming 106 cells have 6.6 ug genome DNA), which calculated as 20ug for genomic DNA PCR. For each sample, at least 5 independent PCR reactions were done, then all the amplicons were pooled together. gRNAs were amplified by PCR (NEB Q5 hot start DNA polymerase) with 28 cycles of reaction using primers targeting U6 promoter and linker between two guide RNAs of each pair. Then the PCR products were second amplified with the Illumina adapter and index sequence. The PCR products were purified with QIAquick PCR purification kit (Qiagen), followed by Illumina deep sequencing.