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| Status |
Public on Jul 08, 2022 |
| Title |
Q107_U |
| Sample type |
SRA |
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| Source name |
Embryo
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| Organism |
Equus caballus |
| Characteristics |
tissue: Embryo
treatment: urea
age: day 14
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| Treatment protocol |
When a 25 ± 3 mm follicle was detected, mares were randomly allocated to a urea (n=9) or control treatment (n=10). The treatment group was fed 0.4 g of feed-grade urea (Hallway Feeds, Lexington, KY) per kg of body weight, mixed with 2.4 kg of sweet feed (Poize 10% crude protein, Hallway Feeds), molasses and mixed grass hay (8.4% crude protein). The control group received identical sweet feed, molasses and hay. Daily meals were divided in two equal amounts given in the morning and afternoon using individual feeding pens. Mares had ad libitum access to water. Mares were artificially inseminated when a 35-mm follicle was determined with 500-million progressively motile sperm pooled together from two fertile stallions. Embryo collection was done at D14 and embryos were preserved in RNAlater (Thermo Fisher Scientific, Waltham, MA) at 4ºC overnight and then kept at -80ºC until RNA isolation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted from embryos using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer’s recommendations. After extraction, RNA concentration and quality were analyzed using a NanoDrop DP-1000 spectrophotometer (Agilent Technologies, Palo Alto, CA) and a Bioanalyzer® (Agilent, Santa Clara, CA). All samples had a 260/280 ratio > 2.0, a 28S:18S rRNA ratios >1.8, and RNA integrity number (RIN) > 8 (8.95 ± 0.4, mean ± SEM). A total of 1 μg of RNA was treated with DNase I (Ambion Inc., Austin, TX) for 30 minutes at 37ºC to remove genomic DNA according to manufacturer’s instructions. The extracted RNA was kept at -20ºC until further analyses.
The mRNA was enriched using oligo(dT) beads. The mRNA was fragmented randomly in fragmentation buffer, followed by first strand cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina, San Diego, CA) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. Double-stranded cDNA was purified using AMPure XP beads (Beckman Coulter, Beverly, CA). In order to select cDNA fragments of 150 base pairs in length, the library fragments were purified with AMPure XP system (Beckman Coulter). The final library was obtained by PCR amplification and purification of PCR products by AMPure XP beads (Beckman Coulter, Beverly, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
The Fastq files were evaluated for read quality using FastQC 0.11.4
Trim Galore 0.4.1 was used for adapter and read quality trimming (Phred score threshold of 30).
Reads were mapped to the Equus caballus reference genome (EquCab 3.0) using the software STAR 2.5.3a (Dobin, Davis et al. 2013)
Reads were annotated with the equine reference annotation from NCBI using Cufflinks 2.2.1
Fragments per kilobase per million (FPKM) were used to determine the expression level of genes
Cuffdiff 2.2.1 was used to calculate differentially expressed genes (DEG) between samples from the control and urea groups
Significance level was set at FDR-adjusted p-value of the test statistic < 0.1 using a Benjamini-Hochberg correction
Genome_build: EquCab 3.0
Supplementary_files_format_and_content: Normalized abundance measurements- Cufflink Gene.FPKM.Tracking
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| Submission date |
Jul 08, 2019 |
| Last update date |
Jul 08, 2022 |
| Contact name |
Barry A. Ball |
| Organization name |
University of Kentucky
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| Department |
Veterinary Scinece
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| Lab |
Reproduction
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| Street address |
108 Gluck Equine Research Center,
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| City |
Lexington |
| State/province |
Kentucky |
| ZIP/Postal code |
40546-0099 |
| Country |
USA |
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| Platform ID |
GPL24409 |
| Series (2) |
| GSE133972 |
EFFECT OF ORAL UREA ADMINISTRATION ON THE TRANSCRIPTOME OF THE EQUINE EMBRYO |
| GSE133974 |
EFFECT OF ORAL UREA ADMINISTRATION ON THE TRANSCRIPTOME OF THE EQUINE ENDOMETRIUM AND EMBRYO |
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| Relations |
| BioSample |
SAMN12232213 |
| SRA |
SRX6416581 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3931518_Q107_U_1.fpkm_tracking.gz |
888.5 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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