Journal: Nature
Article Title: Developmental convergence and divergence in human stem cell models of autism
doi: 10.1038/s41586-025-10047-5
Figure Lengend Snippet: ( 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.
Article Snippet: Then 5 × 10 6 HEK293T were plated onto each of 2 poly-ornithine (5 μg ml −1 ) coated 10-cm plates for 24 h. Media from both plates was replaced with Opti-MEM before cells from 1 10-cm plate were transduced with a lentiviral vector (pLV-KRAB-dCas9 (Addgene no. 71236) or CROP-seq-opti-dsRed (Addgene no. 201999)) while the other plate of cells was transfected with 15 μg of the pooled plasmid library, 15 μg of psPAX2 and 1.5 μg of VSV-G with lipofectamine 3000 following the manufacturer’s instructions.
Techniques: Plasmid Preparation, Flow Cytometry, Expressing, Virus, Single Cell, Sequencing, Control, Whisker Assay
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