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| Status |
Public on May 17, 2016 |
| Title |
MEC_ENDO_Rep2 |
| Sample type |
SRA |
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| Source name |
uterine endometrium
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| Organism |
Sus scrofa |
| Characteristics |
infection status: MEC
tissue: uterine endometrium
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| Treatment protocol |
Samples of endometrium (including adherent placental layers) adjacent to the umbilical attachment site and fetal thymus were snap-frozen in liquid nitrogen and immediately stored at -80°C.
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| Growth protocol |
133 Landrace gilts were obtained from a high-health nucleus herd that was free of PRRSV according to clinical signs and serologic testing. Groups of gilts were selected over 12 bi-weekly replicates, estrus-synchronized, and bred homosperimcally to Yorkshire boars. Pregnant gilts were housed in gestation stalls until gestation day 80±1, and then transferred to a biosafety level 2 facility at the University of Saskatchewan. After a period of acclimation, 114 gilts were inoculated on gestation day 85±1 (Experiment day 0) with 1x105 TCID50 PRRSV isolate NVSL 97-7895: 2 mL intramuscularly and 1 mL into each nostril. Nineteen control gilts were mock inoculated in a similar manner with Minimal Essential Medium (MEM). Gilts were humanely euthanized on Experiment day 21. The gravid uterus of each gilt tract was removed intact, placed in a trough, and orientated from left oviduct to right oviduct. The uterine horns were opened and fetuses numbered sequentially according to their position within the horn. The preservation status of each fetus was recorded in situ as: viable (live at the time of gilt necropsy, normal pink appearance), meconium-stained (live at time of gilt necropsy, stained with meconium), decomposed (dead prior to gilt necropsy, with primarily white skin), autolysed (dead prior to gilt necropsy, with over 50% brown-colored skin), or mummified (dead, <20 cm in length and considered dead prior to PRRSV-inoculation). Mummified fetuses were counted, but excluded from sampling and further analyses. After clamping the umbilical cord, each fetus (starting with live fetuses i.e. those classified as viable or meconium-stained) was removed from the uterus together with its umbilical cord, and a portion of allantochorion and uterus adjacent to the umbilical stump.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 10-20 mg of endometrium and thymus of each fetus using an ‘All Prep RNA/DNA isolation Mini kit’ (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry using a Nanodrop ND 2000 (Thermo Fisher Scientific, Waltham, USA). RNA quality was assessed by digital electrophoresis using a ‘2200 Tapestation’ (Agilent Technologies, Santa Clara, USA). The mean RNA Integrity Number (RIN) sample value was 9.0 for thymus (range 8.0-9.8) and 8.3 for endometrium (range 7.5-8.8).
A total of 96 cDNA libraries were constructed for RNA-sequencing (RNA-seq) from thymus and endometrium samples of individual fetuses. Libraries were constructed from 1 μg of input total RNA using the TruSeq RNA sample preparation kit v2 (Illumina, San Diego, USA) according to the manufacturer’s instructions. DNA libraries were sequenced at the Genome Quebec Innovation Centre (McGill University, Montreal, Canada). Library insert size distribution was assessed on a LabChip GX instrument (Perkin Elmer, Waltham, USA) using a high sensitivity chip. Library concentration was determined using a KAPA Library Quantification Kit (KAPA Biosystems) on a Lightcycler 480 II instrument (Roche Life Science, Indianapolis, USA). Cluster formation was performed on the cBot instrument (Illumina) with eight libraries per lane (7 pM per library) using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Paired-end 100 bp cycle sequencing was performed on a HiSeq 2000 (Illumina) using the TruSeq SBS kit v3-HS (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Base calling: Real-time analysis and base calling were performed using the Hi-seq Control Software, version 2.2.38 (Illumina) to generate sequence reads with associated base quality scores.
Removal of low quality reads: RNA-seq reads, flagged as low quality by CASAVA 1.8 (Illumina), or with an average PHRED read quality score below 15, or in which ≥5 of the last 10 bases had a quality score below 2, were removed.
Sequence alignment: Reads were aligned to the pig reference genome sequence assembly (Sscrofa10.2) using TopHat 1.4.0 with default parameters. The number of reads uniquely mapped to each gene was determined using Ht-seq count (v0.5.3.p3).
Differential gene expression analysis: Differentially expressed genes (DEG) were identified using the Bioconductor package ‘edgeR’. Raw expression counts were converted to counts per million (CPM). Genes with very low expression levels were filtered out of the dataset by setting an expression threshold of CPM>1 in at least 12 samples. Compositional differences between libraries due to difference in library size were normalized using the ‘Trimmed mean of M values’ (TMM) method. A negative binomial model was fitted to the data and gene and tag-wise dispersions were estimated using the quantile-adjusted conditional maximum likelihood (qCML) method. Differential expression was determined using an exact test for the following three contrasts for both tissues: UNINF v CON, INF v UNINF, and MEC v INF. Genes were classified as differentially expressed if they had an absolute fold change >1.5 and a False Discovery Rate (FDR) <0.05.
Weighted Gene Network Correlation Analysis: Gene co-expression network analysis was performed using the software package ‘WGCNA’ for each of the three contrasts in two tissues described above. The normalized CPM expression data generated by ‘edgeR’ were transformed using the function log2 (x+1) to stabilize variance prior to use in WGCNA. To construct the network, a matrix of co-expression similarity, pairwise correlations, for all genes across all samples in each contrast was made. Next, a weighted network adjacency matrix was obtained by raising the co-expression similarity to a power β whose value was determined by application of the approximate scale-free topology criterion [29]. After network construction, average linkage hierarchical clustering was used to identify clusters of highly interconnected gene ‘modules’ using the topological overlap measure of network interconnectedness as the input. A dynamic hybrid tree cutter was used to identify the branches of the hierarchical clustering dendogram corresponding to modules. Network construction and module detection were performed using the function ‘blockwiseModules’, with the following parameter values: a maximum block size of 15000 (sufficiently large to analyze all genes in a single block), a minimum module size of 30, a medium sensitivity to cluster splitting of 2, and a cut height for module merging of 0.25. The value of β ranged from 6-10. The gene expression profile of a module can be summarized by the module ‘eigengene’, defined as the first principal component of the expression matrix. Correlations between the module eigengene and the two categorical traits in each contrast were determined. Correlations between the eigengene and viral RNA titers were also calculated for the appropriate contrasts. The threshold for association significance was set at an absolute correlation of >0.3 and a P value of <0.1.
Genome_build: Sscrofa 10.2
Supplementary_files_format_and_content: Text files that contain the raw sequence counts for each gene (Ensembl gene identifier) produced by the program Ht-seq count.
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| Submission date |
Jul 22, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
James Michael Wilkinson |
| E-mail(s) |
[email protected]
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| Organization name |
University of Alberta
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| Department |
Agriculture, Food, and Nutritional Science
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| Street address |
Ag/For Building
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| City |
Edmonton |
| State/province |
Alberta |
| ZIP/Postal code |
T6G 2P5 |
| Country |
Canada |
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| Platform ID |
GPL11429 |
| Series (1) |
| GSE71205 |
Genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus |
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| Relations |
| BioSample |
SAMN03893113 |
| SRA |
SRX1115548 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1829877_20582_htseq_count.stats.txt.gz |
101.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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