title: GSM8434999: GWAS CRISPR sample4; Homo sapiens; OTHER
library_construction_protocol: EndoC-βH1 cells were infected with lentivirus expressing Zim3 KRAB-dCas9-NLS-P2A-EGFP supplemented with 5 μg/mL polybrene. GFP+ cells were sorted for generating CRISPRi cell line. Then, CRISPRi EndoC-βH1 cells were transduced with a low multiplicity of infection (MOI) of lentivirus containing sgRNA library cassette in order that less than 20% of the cells were BFP+ to ensure the majority cells received only one lentivirus per cell. At 14 days post transduction, the treated cells were sorted using fluorescence activated cell sorting (FACS). Briefly, the sorted cells with greater than 85% viability are qualified for 3' digital gene expression by profiling ~100,000 individual cells per sample. Partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Dual Indexed libraries are generated and sequenced from the cDNA and 10x Barcodes are used to associate individual reads back to the individual partitions. The detailed process according to 10x Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 User Guide with Feature Barcode technology for CRISPR Screening.