superscript iv vilo master mix with ezdnase enzyme reverse transcription kit  (Thermo Fisher)

Journal: Nature Communications

Article Title: Dyna-vivo-seq unveils cellular RNA dynamics during acute kidney injury via in vivo metabolic RNA labeling-based scRNA-seq

doi: 10.1038/s41467-024-54202-4

Figure Lengend Snippet: a UMAP showing PST cells from distinct time points. b UMAP showing the fraction of labeled transcripts per cell in vivo 4sU-labeled PST cells. c UMAP showing H-PST and L-PST cell clusters. UMAP visualization of H-PST cells responded to uni-IRI for distinct time points characterized by conventional splicing-based ( d ) or metabolic labeling-based RNA velocity analysis ( e ). Cells are color-coded by treatment time. The streamlines reveal the integration paths of local projections moving from the observed state to the extrapolated future state. f UMAP visualization of L-PST cells responded to uni-IRI for distinct time points characterized by metabolic labeling-based RNA velocity analysis. g Bar plot showing the expression of Havcr1 in H-PST and L-PST cells at distinct time points. n = 2240 cells for L-PST, n = 973 for H-PST. Data shown as mean ± SD. h Phase portraits showing total-new RNA planes of Havcr1 . i UMAP plots colored by smoothed total RNA level based on local averaging. The regulon activity of transcription factors Jun ( j ) and Cebpb ( k ) along with the AKI progression. In new RNA-based analysis, n = 114, 870, 79, 241, and 251 cells for NC, I_30 min, I_50 min, IR_3.8 h, and IR_5.3 h groups; In old RNA-based analysis, n = 54, 708, 44, 19, and 32 cells for NC, I_30 min, I_50 min, IR_3.8 h, and IR_5.3 h groups. Data shown as mean ± SD. l Signaling pathway role heatmap showing THBS signaling pathway network in Fib, C1q- Macro, C1q+ Macro, and H-PST cells. Source data are provided as a Source Data file.

Article Snippet: The barcoded beads were then resuspended in a reverse transcription mix, consisting of 1× RT buffer, 1 mM dNTP (TransGen Biotech, cat# AD101-12), 2.5 μM Template Switch Oligo (Sangon, 5’-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3’), 1U/μL Recombination RNase Inhibitor (TaKaRa, cat# 2313 A), 10 U/μL reverse transcription enzyme (Thermo Fisher, cat# EP0753), 1 mM GTP (Thermo Fisher, cat# R0461), and 5% PEG-8000 (Beyotime, cat# R0056).

Techniques: Labeling, In Vivo, Expressing, Activity Assay