GSE109660
RNA-sequencing (RNA-seq) of non-manipulated ventricular tissue only of 1- (P1) and 2-day (P2) old neonatal mice
Purpose: The goal of this study is to compare the NGS-derived from ventricular trascriptome profiling (RNA-seq) of 1- and 2-day old mouse hearts to gain insight into the molecular bases responsible for the decline in the heart regeneration competence between P1 and P2 mice Methods: Ventricular cardiac trascriptome profiling were generated by total RNA was extracted from only the ventricles of P1 (n=3) or P2 (n=3) hearts using Quiazol lysis reagent (Quiagen) and RNAeasy extraction kit (Qiagen) according to manufacturer's instructions. Polyadenylated RNA was isolated from 1-5 µg of total RNA using Dynabeads mRNA purification kit (Invitrogen). Polyadenylated RNA was then fragmented, retro transcribed and finally re-synthetized to double strand DNA molecules using DNA polymerase I (Invitrogen). End-pair, A-tailing, adapter ligation and size selection were then performed. RNA-seq analysis was performed using the 50 nt long paired-end reads obtained from sequencing 6 samples of mice heart tissue at two different time points and using three biological replicates for each time point. Firstly, the quality of the reads was verified using FastQC v0.11.21. Then, the reads were mapped to Mus musculus reference genome vGRCm38 2, using tophat v2.0.143. Following mapping, the transcript and normalization counts were performed using cuffdiff v2.2.1 4. The differential expression analysis of normalized counts was performed using cummeRbund v2.12 5, a widely used R v3.2 package which works together with the cuffdiff software; the applied threshold on the parameter alpha in the function getSig of the package was <= 0.05. Results: We found that most differentially-expressed transcripts encode extracellular matrix components and structural constituents of the cytoskeleton. Conclusion: These results suggest that the stiffness of the local microenvironment, rather than cardiac cell-autonomous mechanisms, crucially determines the ability or inability of the heart to regenerate. Overall design: Ventricular mRNA profiles of  (WT) of 1- and 2-day old neonatal mice were generated by deep sequencing, in triplicate, using Illumina HiSeq2500.