Transcriptome of E9.5 WT and Smyd1-KO mouse hearts

Purpose: The goal of this study is to identify the differential cardiac transcriptome profiling between WT and Smyd1 null (Smyd1-KO) hearts at E9.5 using RNA-seq. Methods: mRNA profiles of E9.5 WT and Smyd1-KO mouse hearts were generated by deep sequencing, n=3 for each genotype, using Illumina HiSeq2500. The sequence reads were aligned to the mm10 reference genome using STAR via the bcbio-nextgen RNA-sequencing pipeline. Differential gene expression was determined by DEseq2. Results: 1756 genes were differentially expressed between WT and Smyd1-KO hearts [adjusted P value <0.05, |log2(Fold Change)| > 0.5], with 1130 upregulated and 626 downregulated in E9.5 Smyd1-KO hearts. WT and Smyd1-KO hearts at E9.5, n=3 for each genotype, were dissected in cold PBS and immediately stored in RNAlater (Thermo, AM7020) solution, and were kept in -20C until use. RNA from individual embryonic hearts was extracted using the RNAqueous-Micro total RNA isolation kit (Thermo, AM1931). The concentration and quality of purified RNA were assessed by Tape Station (Agilent). RNA samples with RNA integrity numbers (RIN) ranged from 8.0 to 10.0 were used for library prep and sequencing.研究目的：本研究旨在通过RNA测序（RNA-seq），鉴定胚胎发育第9.5天（E9.5）野生型（Wild Type, WT）与Smyd1基因敲除（Smyd1 null, Smyd1-KO）小鼠心脏之间的差异心脏转录组谱。 实验方法：采用Illumina HiSeq2500平台对E9.5野生型与Smyd1-KO小鼠心脏的mRNA表达谱进行深度测序，每个基因型设置3个生物学重复（n=3）。测序reads通过bcbio-nextgen RNA测序流程，借助STAR软件比对至mm10参考基因组。采用DEseq2进行差异基因表达分析。 实验结果：在E9.5的Smyd1-KO小鼠心脏中，共鉴定出1756个差异表达基因（校正P值<0.05，|log₂(折叠变化)|>0.5），其中1130个基因在Smyd1-KO心脏中表达上调，626个基因表达下调。 分别采集E9.5的野生型与Smyd1-KO小鼠心脏，每个基因型3例，于预冷的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中解剖后，立即置于RNAlater（赛默飞世尔，货号AM7020）溶液中保存，于-20℃冷藏待用。使用RNAqueous-Micro总RNA提取试剂盒（赛默飞世尔，货号AM1931）从单个胚胎心脏中提取总RNA。采用Tape Station（安捷伦）评估纯化后RNA的浓度与质量，选取RNA完整性数（RNA Integrity Number, RIN）介于8.0至10.0之间的RNA样本用于文库构建及测序。