Extended Data Fig. 13: M5 Regulator CRISPRi methods and validation.
From: Developmental convergence and divergence in human stem cell models of autism
(a) Schema of CROP-Seq vector. (b) Example of gating for flow cytometry. Left shows a set of hNPCs not expressing virus used for gating compared with hNPCs expressing both dCas9-Krab (GFP) and Crop-Seq-opti-dsRed (TdTomato). Box in the upper right shows double positive cells selected for downstream analysis (~20% of live cell fraction). (c) QC measures for CRISPRi single cell libraries organized by gRNA negative (no gRNA detected in sequencing), Control gRNA positive, or Target gRNA positive. (d) Number of gRNA UMIs per cell. X-axis shows cut-off of 10 UMIs used to consider a cell gRNA positive (left). Number of of unique gRNAs expressed in each cell (x-axis). Only cells expressing gRNA for a single gene-target are retained for downstream analyses. (e) Single cell UMAPs of markers used to differentiate cycling and non-cyling NPCs (f) Single cell UMAP coloured by Target-gene presence (g) Proportion of cells uniquely expressing gRNAs barcodes for each target within each experiment (n = 18 per Gene comprised of 6 technical replicates, 3 gRNAs barcodes per target, for Controls n = 108: 18 gRNAs across 6 technical replicates). TP53 show increased proportion compared to non-targeting controls (NTCs). Boxplots a-c show: centre, median, lower hinge – 25% quantile, upper hinge – 75% quantile, lower whisker – smallest observation greater than or equal to lower hinge –1.5× interquartile range, upper whisker – largest observation less than or equal to upper hinge +1.5× interquartile range. ***Family wise corrected p < 0.005, Dunnets post-hoc contrast of linear model.
