title: GSM5554052: A375_Day35_PP020_CasRx_nls_R1; Homo sapiens; OTHER
library_construction_protocol: The regions encompassing the sgRNAs were firstly PCR-amplified for around 20-23 cycles . The second round of PCR were employed to attach the illumina adaptors and index for around 10-12 cycles. These PCR products were gel purified and pooled for high-throughput sequencing to identify sgRNA abundance on illumina PE150 sequencing platform (Novogene, China). The pooled synthesized oligos (Synbio Technologies, China) were PCR amplified and then cloned into pLentiCasRxDR-puro vector (for expressing CasRx sgRNA) via BsmBI site by Gibson Assembly. The ligated Gibson Assembly mix was transformed into self-prepared electrocompetent Stable E. coli cells by electro-transformation to reach the efficiency with at least 100X coverage representation of each clone in the designed library. The transformed bacteria were cultured directly in liquid LB medium for 16~20 hours at temperature 30°C to minimize the recombination events in E. coli. The library plasmids were then extracted with EndoFree Maxi Plasmid Kit (TIANGEN, Cat no. 4992194). Ensembl gene and lncRNA annotations (version: GRCh38) are used to extract the sequences of corresponding genes or lncRNAs. For genes (or lncRNAs) with multiple transcripts, the transcripts that have the corresponding RefSeq ID are used. We first enumerate all possible guides (22bp) that span the entire transcript, then remove guides that (1) map to more than one location in the human genome and transcriptome (allowing up to 1 mismatch) or (2) contains BsmBI digestion sites (“CGTCTC” or “GAGACG”). The remaining guides are randomly selected if they hit the greatest number of transcripts (“common” guides) within the same gene/lncRNA. If all the guides that hit the greatest number of transcripts are selected, the remaining guides that hit the second greatest number of transcripts will be randomly selected, and so on. For essential and non-essential genes, 35 guides are designed per gene. For other genes or lncRNAs, 15 guides are designed per gene or lncRNA.