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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 13, 2019 |
| Title |
RNA sequencing identifies differentially expressed genes in embryonic cardiomyocytes following knockdown of DNMT1 expression for 72 h |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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 37°C 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.
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| Overall design |
The mRNA profile in embryonic cardiomyocytes with knockdown of DNMT1 expression was generated by deep sequencing (n=3 for negative siRNA and n=3 for DNMT1 siRNA), using Illumina NextSeq500.
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| Contributor(s) |
Fang X, Rivkees SA, Wendler CC |
| Citation(s) |
34235895 |
| Submission date |
May 13, 2016 |
| Last update date |
Aug 17, 2021 |
| Contact name |
Xiefan Fang |
| E-mail(s) |
xiefanfang@ufl.edu
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| Organization name |
University of Florida
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| Department |
Pediatrics
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| Street address |
1200 Newell Drive, ARB R1-148
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| City |
Gainesville |
| State/province |
Florida |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA321550 |
| SRA |
SRP075146 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE81446_RAW.tar |
980.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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