PLX040787

GSE81446: RNA sequencing identifies differentially expressed genes in embryonic cardiomyocytes following knockdown of DNMT1 expression for 72 h

• Organsim mouse
• Type RNASEQ
• Target gene
• Project ARCHS4

Purpose: This study aims to identify the differentially expressed genes in embryonic cardiomyocytes following the knockdown of DNMT1 expression. Methods: Primary cardiomyocytes were isolated from mouse embryonic day (E) 13.5 ventricles, and cultured for 48 h at 37C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT1 siRNA (#GS13433) or AllStars negative control siRNA (#1027281) at 12 nM using lipofectamine RNAiMAX (n=3 per treatment). At 72 h after transfection, cells were released with Accutase (Innovative Cell Technologies, San Diego, CA) and dissociated with Accumax (Innovative Cell Technologies). To sort cardiomyocytes, dissociated cells were stained with anti-VCAM1 antibody conjugated with allophycocyanin (APC; BioLegend, San Diego, CA) and magnetically sorted using anti-APC microbeads and magnetic assisted cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA was isolated from the sorted cardiomyocytes with the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA). RNA-Seq libraries were prepared with the NEBNext mRNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (NEB, Ipswich, MA). The Illumina-adapted libraries were pooled at equal molar ratio and sequenced with one High Output 1x75 cycles run on a NextSeq500 sequencer (Illumina). The fastq files generated from RNA-Seq were uploaded to the UF Research Computing Galaxy instance developed by the University of Florida. The data were cleaned with the following steps: removed sequencing artifacts, trimmed 5 or 3 ends with low scores, removed adaptor contamination, and filtered low quality reads by the FastQC program. The remaining high quality RNA-Seq reads were mapped to the mouse genome (mm10) with the Tophat2 tool. Counting of RNA-seq reads were performed with HTSeq. Differential expression (DE) of genes between treatments was analyzed using two methods: R packages EdgeR and DEseq2, with Ensembl Mus_GRCm38.79.gtf as the reference annotation. Genes with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Unique DE genes were identified by combining the results generated from the two analytical methods. Results: DE analysis by DEseq2 and EdgeR software revealed that 801 genes were up-regulated, and 494 genes were down-regulated after knockdown of DNMT1 expression for 72 h. SOURCE: Xiefan Fang (xiefanfang@ufl.edu) - University of Florida