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| Status |
Public on Sep 18, 2019 |
| Title |
K130_UREA |
| Sample type |
SRA |
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| Source name |
Uterus
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| Organism |
Equus caballus |
| Characteristics |
tissue: Endometrium
treatment: urea
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| Treatment protocol |
Mares received a urea-treatment or control infusion (n = 7 mares/group) in a crossover design. The intervening estrous cycle was skipped and served as a washout cycle. Mares received 2500 IU of human chorionic gonadotropin (hCG) (Chorulon; Intervet, Millsboro, DE) intravenously when they had a follicle of at least 35 mm in diameter and pronounced uterine edema. Animals were scanned daily by ultrasound for ovulation detection (Day 0 = ovulation) and infusions were initiated at Day 7 of diestrus (D7). On the day of infusion, both jugular veins were catheterized using. Treatment consisted of a loading dose of 0.03 g/kg of urea (Sigma-Aldrich Company, St. Louis, MO) diluted in 100 mL of saline solution (Hospira, Inc, Lake Forest, IL) (15 g of urea for a 500 kg horse) to achieve a rapid increase in urea concentrations, and control mares received 100 mL of saline solution. Subsequently, mares received a bolus injection of urea (0.03 g/kg/hr) diluted in 15 mL of saline solution every 30 minutes over 6 hours (90 g of urea during 6 hours for a 500 kg horse). The control group received the same amount of saline solution as the urea group, 100 mL of saline solution as a loading dose and 15 mL of saline solution every 30 minutes over 6 hours. A uterine biopsy was collected from the base of the uterine horn.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted from endometrial samples 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 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 37℃ to remove genomic DNA according to manufacturer’s instructions.
The extracted RNA (1ng), was used for library preparation and RNA Sequencing. Paired-end reads with 150 nucleotides in length were produced. The RNAseq libraries were prepared with Illumina's TruSeq Stranded mRNAseq Sample Prep kit (Illumina, San Diego, CA). Read 1 aligns to the antisense strand and Read 2 aligns to the sense strand. The libraries were quantitated by qPCR and sequenced on one lane for 101 cycles from each end of the fragments on a HiSeq 4000 using a HiSeq 4000 sequencing kit version 1. The lane produced a total of 700 million reads. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina).
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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 |
May 28, 2019 |
| Last update date |
Sep 18, 2019 |
| 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 (1) |
| GSE131810 |
A high protein model alters the endometrial transcriptome of mares |
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| Relations |
| BioSample |
SAMN11872396 |
| SRA |
SRX5907607 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3820041_K130_UREA.fpkm_tracking.gz |
930.8 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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