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| Status |
Public on Oct 31, 2022 |
| Title |
K19005695 |
| Sample type |
SRA |
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| Source name |
Uterus
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| Organism |
Equus caballus |
| Characteristics |
tissue: Chorioallantois
group: Cluster 4
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from tissue samples using RNeasy Mini Kit (#74104: Qiagen). RNA concentrations and quantities were confirmed using the Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, MA, USA). Then, RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). All samples had a 260/280 ratio >2.0 and RNA integrity number (RIN) >7.0.
A total of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M- MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The Fastq files were evaluated for read quality using FastQC 0.11.8
Trim Galore 0.4.3 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 STAR 2.7.2a (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.fpkm_tracking) were used to determine the expression level of genes
Based on fragments per kilobase of exon (FPKM.fpkm_tracking), all 37 samples were divided into four groups using k-means clustering (R Ver 3.1)
Cuffdiff 2.2.1 was used to calculate differentially expressed genes (DEGs) between samples from the each clusters and control groups
Significance level was set at FDR-adjusted p-value of the test statistic < 0.05 using a Benjamini-Hochberg correction
Genome_build: EquCab 3.0
Supplementary_files_format_and_content: Normalized abundance measurements- Cufflink Gene.FPKM.fpkm_tracking.Tracking
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| Submission date |
Oct 26, 2021 |
| Last update date |
Nov 01, 2022 |
| Contact name |
Harutaka Murase |
| E-mail(s) |
sakuheki@gmail.com
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| Organization name |
Japan Racing Association
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| Street address |
535-13, Nishicha
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| City |
Urakawa |
| State/province |
Hokkaido |
| ZIP/Postal code |
0570171 |
| Country |
Japan |
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| Platform ID |
GPL26749 |
| Series (1) |
| GSE186617 |
Transcriptomic analysis of the chorioallantois in equine premature placental separation. |
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| Relations |
| BioSample |
SAMN22575604 |
| SRA |
SRX12780499 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5657871_K19_5695_FPKM.fpkm_tracking.gz |
808.8 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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