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| Status |
Public on Feb 09, 2017 |
| Title |
RNA sequencing identifies differentially expressed genes in adult left ventricles following in utero caffeine exposure during embryonic day 6.5-9.5 |
| 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 adult left ventricles treated with caffeine during early embryogenesis. Methods: Pregnant CD-1 mice were treated with vehicle (normal saline) or 20 mg/kg caffeine via i.p. injection once daily during embryonic day (E) 6.5-9.5. Pups were born and raised to one year of age. Left ventricles from adult male mice were dissected and used for total RNA isolation with the RNeasy Plus Mini kit (Valencia, CA, USA). mRNA was isolated from total RNA using NEXTflex™ Poly(A) Beads (Bioo Scientific, Austin, TX, USA). Sequencing libraries were prepared with the NEBNext® mRNA Library Prep Master Mix Set for Illumina (NEB, Ipswich, MA, USA) and the NEBNext Multiplex Oligos for Illumina (NEB). Illumina-adapted libraries (n=3/treatment) were pooled at equal molar ratio and sequenced with one High Output 1x75 cycles run on a NextSeq500 sequencer (Illumina, San Diego, CA, USA). 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 R package EdgeR, 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. Results: DE analysis revealed that 85 genes were up-regulated, and 31 genes were down-regulated in the adult left ventricles treated with in utero caffeine.
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| Overall design |
mRNA expression profiles in the adult left ventricles treated with vehicle or 20 mg/kg caffeine during E6.5-9.5 were generated by deep sequencing (n=3/treatment), using Illumina NextSeq500.
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| Contributor(s) |
Fang X, Rivkees SA, Wendler CC |
| Citation(s) |
27677355 |
| Submission date |
Mar 08, 2016 |
| Last update date |
May 15, 2019 |
| 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 |
PRJNA314693 |
| SRA |
SRP071312 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE79013_RAW.tar |
920.0 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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