pucmini icap php eb  (Addgene inc)

Journal: bioRxiv

Article Title: Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

doi: 10.64898/2026.03.16.711480

Figure Lengend Snippet: ( A ) Schematic of mouse whole brain in vivo Perturb-seq using 10x Genomics Flex Apex platform. ( B ) UMAP of whole brain in vivo Perturb-seq dataset encompassing 7.7 million sequenced nuclei, colored by developmental neighborhoods, anatomical region, and neurotransmitter type, inferred using MapMyCells . ( C ) Heatmap of the gene expression levels of 1,947 neurodevelopmental disease-associated risk genes in non-targeting control nuclei across different developmental neighborhoods. ( D ) Histogram of in vitro gRNA activity distribution of 45 selected gRNAs (15 genes, 3 gRNAs per gene) compared to safe-targeting controls by insertion-deletion analysis. ( E ) Immunofluorescence image of sagittal section of a P37 mouse brain retro-orbitally administered with 6e8 total vg per gram of body weight of AAV PHP.eB encoding either GFP or mScarlet (1:1 ratio) (scale bar, 1 mm), accompanied by zoomed in images to show representative MOI in each major brain region (scale bar, 50 μm), and stacked bar plot quantifying GFP and mScarlet viral labeling efficiency as well as double labeling rate. ( F ) UMAPs of whole brain in vivo Perturb-seq dataset separated by neighborhoods, colored by inferred cell subclass using MapMyCells . ( G ) Violin plots of number of genes and RNA UMIs recovered per nucleus from each developmental neighborhood. ( H ) Ranked bar plot showing proportion of sampled nuclei by brain region. ( I ) Stacked bar plot showing percentage of nuclei with no guide, single, double, or multiple guide assignment within each developmental neighborhood. ( J ) Histogram of total nuclei number distribution of nuclei recovered per perturbation. ( K ) Ranked dot plot of nuclei number in each perturbation and cell type pair and the minimum cell number cut off for perturbation and cell type pair for downstream analyses (dashed line). ( L ) Scatter plot of weighted mean log fold-changes of target genes across all cell types against their weighted mean expression levels in non-targeting control nuclei.

Article Snippet: Briefly, HEK293T cells were transfected with above pooled plasmid library or plasmid encoding a PiggyBac transposase , along with AAV-PHP.eB capsid (Addgene #103005) and pHelper plasmids using PEI (Polysciences, #24765-1) at 80-90% confluency.

Techniques: In Vivo, Gene Expression, Control, In Vitro, Activity Assay, Immunofluorescence, Labeling, Expressing

Journal: bioRxiv

Article Title: Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

doi: 10.64898/2026.03.16.711480

Figure Lengend Snippet: (A) Immunofluorescence image of sagittal section of a P37 mouse brain retro-orbitally injected at P16 with high (1e9), mid (6e8), or low (1.5e8) total vg per gram of body weight of AAV PHP.eB encoding either GFP or mScarlet (1:1 ratio) (scale bar = 1 mm). (B) Quantification of GFP and mScarlet viral labeling efficiency as well as double labeling rate in (A). (C) Representative FACS gating strategy to enrich transduced neuronal nuclei. (D) Bar plot of sex and weight at harvest of animals used in this study. (E) Animal tracking information showing the litter, age at harvest for each animal, as well as AAV-labeling rate by FACS and total nuclei number per hemisphere used for Flex hybridization. (F) Schematic of snRNA-seq data processing and quality control workflow.

Article Snippet: Briefly, HEK293T cells were transfected with above pooled plasmid library or plasmid encoding a PiggyBac transposase , along with AAV-PHP.eB capsid (Addgene #103005) and pHelper plasmids using PEI (Polysciences, #24765-1) at 80-90% confluency.

Techniques: Immunofluorescence, Injection, Labeling, Hybridization, Control