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. 2018 Dec 4:5:180287.
doi: 10.1038/sdata.2018.287.

Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs

Isabela Tiemy Pereira  1 , Lucia Spangenberg  2 , Anny Waloski Robert  1 , Rocío Amorín  2 , Marco Augusto Stimamiglio  1 , Hugo Naya  2 , Bruno Dallagiovanna  1
Affiliations

Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs

Isabela Tiemy Pereira et al. Sci Data. .

Abstract

The regulation of gene expression acts at numerous complementary levels to control and refine protein abundance. The analysis of mRNAs associated with polysomes, called polysome profiling, has been used to investigate the post-transcriptional mechanisms that are involved in different biological processes. Pluripotent stem cells are able to differentiate into a variety of cell lineages, and the cell commitment progression is carefully orchestrated. Genome-wide expression profiling has provided the possibility to investigate transcriptional changes during cardiomyogenesis; however, a more accurate study regarding post-transcriptional regulation is required. In the present work, we isolated and high-throughput sequenced ribosome-free and polysome-bound RNAs from NKX2-5eGFP/w HES3 undifferentiated pluripotent stem cells at the subsequent differentiation stages of cardiomyogenesis: embryoid body aggregation, mesoderm, cardiac progenitor and cardiomyocyte. The expression of developmental markers was followed by flow cytometry, and quality analyses were performed as technical controls to ensure high quality data. Our dataset provides valuable information about hESC cardiac differentiation and can be used to investigate genes potentially controlled by post-transcriptional mechanisms.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Cardiomyogenic differentiation of hESCs.
(a) Schematic representation of the steps followed for RNA-seq data generation. (b) Schematic representation of the cardiomyogenic differentiation protocol, indicating days of differentiation and timing of specific induction. (c) Representative images of EBs during differentiation showing NKX2-5/eGFP expression on D15. Phase contrast (PC) and eGFP fluorescence (left image), eGFP fluorescence (right image). 250 μm scale. (d) Representative images of differentiated cardiomyocytes stained for cTnI on D20. Isotype control (left image), cTnI staining (right image). White rectangle as 50 μm scale.
Figure 2
Figure 2. Expression of markers followed during cardiomyogenic differentiation.
Flow cytometry analysis of (a) D3 and D4 (CD56), (b) D3, D9 and D15 (eGFP) and (c) D15 (cTnT) differentiating cells. Representative dot plots (n = 3). Non-induced (ni) cells were used as a control for differentiation. (d) Quantification of percentage of positive cells for the indicated markers (n = 3) (Data Citation 1).
Figure 3
Figure 3. Polysome profiling followed by RNA-seq during cardiomyogenic differentiation.
(a) Schematic representation of the sucrose gradient used to segregate ribosome-free and ribosome-bound RNAs and representative polysome profile (hESCs - D0 replicate 2) recorded at 254 nm. Ribosome-free and polysome fractions are indicated. (b-d) Representative quality analysis of ribosome-free and polysome-bound samples. (a) RNA quality analysis using Agilent 2100 Bioanalyzer. (b) cDNA library quality analysis using Agilent 2100 Bioanalyzer. (c) RNA-sequencing reads quality analysis using FastQC. All representative images correspond to D0 hESC sample, replicate 2.
Figure 4
Figure 4. Data quality analysis of RNA-seq and validation.
Principal component analysis (PCA) of (a) all sequenced samples (total 30 samples), (b) ribosome-free and (c) polysome-bound samples at D0, D1, D4, D9 and D15 (n = 3). RPKM values (polysome-bound) heatmap of (d) developmental markers showing mesoderm, cardiac mesoderm, endoderm and ectoderm genes expression; and (e) cardiac markers showing cardiomyocytes (CM), endothelial cells (EC) and smooth muscle cells (SMC) genes expression. (f) RNA-seq data validation of cardiac developmental marker gene expression using qPCR. Relative expression to hESC (D0) of pluripotency (POU5F1 and NANOG), mesoderm (T and EOMES) and cardiac markers (GATA4, NKX2-5 and TNNT2) on distinct days of cardiac differentiation using RNA-seq and q-PCR data. Values are expressed in log2 base.
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References

Data Citations

    1. Robert A., Stimamiglio M. 2018. FlowRepository. FR-FCM-ZYX8
    1. 2018. NCBI Sequence Read Archive. SRP150416
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