Journal: Nature Communications
Article Title: MicroRNAs promote skeletal muscle differentiation of mesodermal iPSC-derived progenitors
Figure Lengend Snippet: RNA-seq after miRNA cocktail exposures shows myogenic-specific comittment enhancement of transcriptional profile. a PC analysis of a MAB-MiPs samples reveals stage-specific, progeny-specific clustering. b Unbiased clustering of MAB-MiPs analyzed by RNA-seq. c – f Volcano plots of fold change vs p -value of all DE genes in different conditions (threshold, P = 0.05, dashed line). MAB-MiPs untreated vs MAB-MiPs+AMC c , MAB-MiPs untreated vs MAB-MiPs+PMC d , fibroblast-MiPs untreated vs fibroblasts-MiPs+AMC e , fibroblast-MiPs untreated vs fibroblasts-MiPs+PMC f . Left side of all charts, all genes and highlighted candidates enriched in untreated-MiPs; right side, genes and candidates enriched in treated-MiPs. Blue dots depict genes that were found downregulated while red dots depict genes that were found upregulated upon treatments. g Quantitation of results obtained from bisulfite sequencing of upstream CpG islands of selected anti-myogenic and pro-myogenic gene pools in treated and untreated cells. Data is depicted as heatmap of average percentages of methylated CpGs. h Quantitation of results obtained from ChIP-qPCR experiments analyzing fibroblast- and MAB-MiPs treated and untreated. H3k9me3, repressive marker; H3k4me2, H3k27ac, permissive markers. Data is depicted as heatmap of average percentages of IP-enriched vs total input DNA ( n = 3). f-, fibroblast-derived. AMC, anti-myogenic cocktail (miRNA-based), PMC, pro-myogenic cocktail (miRNA-based)
Article Snippet: Gene qPCR was performed on 1:5 diluted cDNA obtained from 1 μg total RNA (SybrGreen mix, SSIII cDNA production kit and RNA extraction kit from Thermo Fisher Scientific), using Viia7 384-plate reader (Thermo Fisher Scientific; final primer concentration, 100 nM; final volume, 10 μl; PGK , internal reference; thermal profile, 95 °C 15 s, 60 °C 60 s, 40×). miRNA qPCR was performed on 1:15 diluted cDNA obtained from 20 ng total miRNA preparation (miRNA isolation kit, Thermo Fisher Scientific).
Techniques: RNA Sequencing Assay, Quantitation Assay, Methylation Sequencing, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Marker, Derivative Assay