Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) screens Endoc-betaH1 cell line targeting 61 T2D coding variants genes and 40 RQC function genes.
Two set of genes were selected for Perturb-seq experiments. sgRNAs targeting each gene were selected according to previously published algorithm and cloned into the lentivirus construct containing sgRNA cassette available for 3' direct capture Perturb-seq: hU6-gRNA with CR1CS1-EF1a-BFP. Overall design: 1) Lentiviral production.HEK293T cells were transfected with lentiviral backbone encoding CRISPRi effector or sgRNA library and two other helper plasmids (psPAX2 and pMD2.G). Forty-eight hours later, viral supernatant was harvested and filtered through a 0.45 µm cellulose acetate filter and frozen at -80°C prior to transduction.2) 3' direct-capture Perturb-seq.EndoC-ßH1 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-ßH1 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) and processed according to 10x Genomics Chromium Single Cell 3' Reagent Kits v3.1 User Guide with Feature Barcode technology for CRISPR Screening.