Supplementary Figure 5: Optimization of additional guide constant regions to enable dual-guide 3’ direct capture Perturb-seq.
From: Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing
a) Gaussian kernel density estimates of normalized flow-cytometry measurements representing GFP expression demonstrate CRISPRi activity of sgRNAs with indicated constant regions (programmed with an identical GFP targeting region). Data was collected in three independent biological replicates and a representative replicate is shown. In this experiment, sgRNAs were expressed from a single-guide vector. As all CR2 and CR3 sgRNA variants were highly active, we used CR3 with cs1 in the stem loop (CR3cs1) and CR2 with cs2 at the 3’ end (CR2cs2) for downstream experiments. AU, arbitrary units. b) Median index (guide) UMIs per cell for each of n=45 sgRNAs where grey lines indicate medians. Data are from our dual-guide genetic interaction 3’ direct capture Perturb-seq experiments. c) Median index (guide) UMIs per cell for each of n=45 sgRNAs where grey lines indicate medians. Data are from our dual-guide genetic interaction 3’ direct capture Perturb-seq experiment with an mU6-CR3cs1-hU6-CR1cs1 design. d) Fraction of cells in each cell cycle phase across cells with the indicated perturbations.
