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Series GSE77514 Query DataSets for GSE77514
Status Public on Sep 08, 2017
Title RNA sequencing identifies differentially expressed genes in embryonic cardiomyocytes following knockdown of DNMT3a or DNMT3b expression
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary Purpose: This study aims to identify the differentially expressed genes in embryonic cardiomyocytes following the knockdown of DNMT3a or DNMT3b expression. Methods: Primary cardiomyocytes were isolated from mouse embryonic day (E) 13.5 ventricles, and cultured for 48 h at 37°C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT3a siRNA (#GS13435), DNMT3b siRNAs (#GS13436), or AllStars negative control siRNA (#1027281) at 0.012 pmol 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 nine 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 524 genes were up-regulated, and 586 genes were down-regulated after knockdown of DNMT3a expression for 72 h. With the same analytical methods and criteria, the results of DE analysis showed that the knockdown of DNMT3b increased the expression of 655 genes and decreased the expression of 378 genes.
Overall design mRNA profiles in embryonic cardiomyocytes with knockdown of DNMT3a or DNMT3b expression were generated by deep sequencing (n=3 for negative siRNA, n=3 for DNMT3a siRNA, n=3 for DNMT3b siRNA), using Illumina NextSeq500.
Contributor(s) Fang X, Rivkees SA, Wendler CC
Citation(s) 27306334
Submission date Feb 03, 2016
Last update date May 15, 2019
Contact name Xiefan Fang
Organization name University of Florida
Department Pediatrics
Street address 1200 Newell Drive, ARB R1-148
City Gainesville
State/province Florida
ZIP/Postal code 32610
Country USA
Platforms (1)
GPL19057 Illumina NextSeq 500 (Mus musculus)
Samples (9)
GSM2053527 negative_siRNA_rep1
GSM2053528 negative_siRNA_rep2
GSM2053529 negative_siRNA_rep3
BioProject PRJNA310690
SRA SRP069225

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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE77514_RAW.tar 1.4 Mb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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