Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Figure Lengend Snippet: Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P
Article Snippet: DAPI-negative and MitoTracker-positive living cells were either sorted into TRIzol reagent (Thermo Scientific) for bulk mRNA sequencing or into 384-well plates containing 96 or 384 unique molecular identifier barcode primer sets, ERCC92 spike-ins (Agilent) and dNTPs (Promega) for single-cell mRNA-sequencing (SORT-seq) ( ) using a flow sorter (FACSAriaII, FACSFusion, or FACSJazz; all BD).
Techniques: Mouse Assay, Isolation, Amplification, Sequencing, Expressing, Staining, Flow Cytometry, Cytometry, Construct