Extended Data Fig. 1: MitoPerturb-Seq and quality-control steps.
(A) Schematic of the CROPseq-RFP lentiviral cassette showing transcript expression and poly-adenylation of a gRNA-containing transcript following genomic integration. (B) Flow cytometry plots confirming genome editing efficiency in transgenic Cas9-expressing MEF clones. Clonal cells stably expressing eGFP were transduced with CROPseq-RFP lentivirus carrying two separate gRNAs targeting the eGFP sequence. RFP-high cells displayed loss of eGFP fluorescence, confirming effective CRISPR-Cas9 editing of the target gene. (C) Single-cell heteroplasmy measurements from five Cas9 transgenic MEF clones, six cells per clone. Clone 9, used for MitoPerturb-Seq experiments, is highlighted in red. Data presented as mean values ± SD. (D) Flow cytometry plot showing RFP expression 10 days post-transduction with the pooled CROPseq-RFP gRNA library. Transduction efficiency of 35.5% (MOI = 0.35) indicates that the majority of cells received a single viral particle. (E,F) Bright field images of unfixed/unpermeabilized (E) or fixed/permeabilized (F) MEFs stained with trypan blue. Viable cells (E) have not taken up the dye and remain bright with a well-defined membrane, while permeabilized cells (F) have taken up the dye, with darker-staining nuclei. Average cell size, calculated by the Countess cell counter, is indicated. (G) QC violins from Seurat for RNA-seq and ATAC-seq, dashed red lines indicate thresholds used for filtering, as described in the methods. Outliers with high y-axis values have been removed from selected plots: Total RNA Counts, 27 outliers; Percentage of Mitochondrial Genes, 2 outliers; Total ATAC Counts, 47 outliers; TSS Enrichment 6 outliers. (H-J) QC plots of nCountRNA (H), nCountATAC (I) and nFeatureRNA (J) versus nFeatureRNA (H) or nFeatureATAC (I,J), colored by percent mitochondrial reads, with lines indicating cutoff thresholds shown in violin plots in (G). (K-M) UMAPs showing MitoPerturb-Seq cells, clustered based on overall RNA-seq (K), ATAC-seq (L) or by Weighted Nearest Neighbor (WNN) of combined RNA- and ATAC-seq (M) data, prior to regression of cell cycle heterogeneity. Each point represents an individual cell. Cells are colored by cell cycle phase. Figure created in BioRender. Van den Ameele, J. (2026) https://BioRender.com/j3ro7vq.
