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Fig. 2 | Genome Biology

Fig. 2

From: Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

Fig. 2

Transcriptomic landscape of live, dead, and dying cells. a FACS analysis showing gating strategy for untreated, live cells (PI−/annexin V−) or TNFα-treated dying cells (PI/annexin V+) and dead cells (PI+/annexin V+). b PCA projection of the three cell conditions showing approximate segregation of cell status along the first principal component (PC1), with live and dying cells enriched at lower PC1 values and dead cells enriched at higher values. c PCA projection colored by the percentage mitochondrial genes (“% transcriptome mitochondrial”) shows significant increase along the PC1. d Dead cells exhibit significantly higher percentage of the transcriptome as mitochondrial compared to both live and dying cells. e Unsupervised clustering of the gene expression profiles clusters the cells into three groups, approximately tracking both PC1 of the data and the percentage of transcriptome mitochondrial. f The composition of each cluster demonstrates that cluster 1 is primarily composed of live cells and cluster 2 a mix of live, dying, and dead cells, while cluster 3 is composed mainly of dead cells. g The percentage of transcriptome mitochondrial is significantly different between the three clusters, with a step increase in proportion moving from cluster 1 to 2 and 2 to 3. h Cluster 2 significantly upregulates the MHC class I gene set, suggesting it represents stressed or pre-apoptotic cells. i Differential expression analysis of transcriptomically “healthy” cells within cluster 1 reveals residual differences between cells sorted as live and dead. j The distribution of absolute effect sizes (log fold change) of live vs. dead cells within cluster 1 (x-axis) compared to between clusters 1 and 2 (y-axis) demonstrates the residual effect on the transcriptome of being live/dead sorted is small compared to the inter-cluster expression variance

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