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 ÎŒg 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. Ventricular mRNA profiles of (WT) of 1- and 2-day old neonatal mice were generated by deep sequencing, in triplicate, using Illumina HiSeq2500.研究目的：本研究旨在对比出生1天（P1）与出生2天（P2）小鼠心脏的心室转录组测序（RNA-seq）所得的下一代测序（Next-Generation Sequencing, NGS）数据，以解析P1至P2小鼠心脏再生能力下降的分子机制。 研究方法：通过仅从P1（n=3）或P2（n=3）小鼠心脏心室中提取的总RNA，采用Qiagen公司的Quiazol裂解试剂（Quiazol lysis reagent）与RNAeasy提取试剂盒（RNAeasy extraction kit），严格遵循厂商说明书操作，制备得到心室心脏转录组分析文库。随后，取1~5 μg总RNA，使用Invitrogen公司的Dynabeads mRNA纯化试剂盒（Dynabeads mRNA purification kit）分离聚腺苷酸化RNA。将分离得到的聚腺苷酸化RNA进行片段化处理，经反转录后，借助Invitrogen公司的DNA聚合酶I（DNA polymerase I）合成双链DNA分子。后续依次进行末端修复、A尾加尾、接头连接及片段大小筛选。本研究针对两个时间点的小鼠心脏组织样本共设置6份测序样本，每个时间点设置3次生物学重复，通过测序获取50 nt长度的双端读段（paired-end reads），以此开展RNA-seq分析。首先使用FastQC v0.11.21对读段质量进行质控；随后采用TopHat v2.0.143将读段比对至小鼠参考基因组vGRCm38 2；比对完成后，使用Cuffdiff v2.2.1 4完成转录本定量与标准化计数；基于标准化计数的差异表达分析，采用广泛应用的R v3.2工具包cummeRbund v2.12 5完成，该工具包可与Cuffdiff软件协同工作；分析时将该工具包getSig函数中的alpha参数阈值设置为≤0.05。 研究结果：本研究发现，大部分差异表达转录本（differentially-expressed transcripts）编码细胞外基质（extracellular matrix）成分与细胞骨架（cytoskeleton）结构蛋白。 研究结论：上述结果表明，心脏再生能力的有无主要由局部微环境的刚度决定，而非心肌细胞的自主调控机制。本研究通过Illumina HiSeq2500平台对1日龄与2日龄野生型（WT）新生小鼠的心室mRNA表达谱进行深度测序，并设置3次生物学重复。