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| Status |
Public on Mar 31, 2017 |
| Title |
5072-7DPI |
| Sample type |
SRA |
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| Source name |
7 DPI_whole blood
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| Organism |
Sus scrofa |
| Characteristics |
animal id: 5072
gender: M
days post infection: 7 DPI
wur marker type: AA
viremia level: 5.98129
body weight (kg): 7.80179
tissue: peripheral blood
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Three mL of whole blood were collected into the Tempus Blood RNA tubes (Life Technologies, Carlsbad, CA, USA). Total RNA of trial 3 and 5 was isolated using the Tempus Spin RNA isolation Kit (Life Technologies, Carlsbad, CA, USA) and MagMax for Stabilized Blood Tubes RNA Isolation Kit for Tempus tubes (Life Technologies), respectively, according to the respective manufacturer's instructions. RNA concentration was quantified using a ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA) and RNA quality was assessed using either Agilent 2100 Bioanalyzer or 2200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA, USA). The globin transcripts (HBA and HBB) were reduced using an RNase H based globin reduction method 24 and RNA quality was assessed again.
Poly (A)+ fractions from the globin depleted RNA samples (1.0 µg RNA each) were purified by oligo-dT purification beads (Illumina, Inc., San Diego, USA) and then used to construct cDNA libraries following the TruSeq RNA Sample Preparation Guide (Illumina, Inc., San Diego, USA). Sequencing was performed on HiSeq 2000 System (Illumina, Inc.) using the TruSeqTM Pair End (PE) 100bp Kit (Illumina, Inc.).
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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 |
Sequence reads with base quality scores were produced by the Illumina sequencer. RNA-Seq reads flagged as low quality by the chastity filter in CASAVA 1.8 were removed. In addition, we removed reads with an average read quality score below 15 and reads in which over 5 of the last 10 bases had a PHRED quality score below 2.
Sequence reads were aligned to pig reference genome sequence assembly (Sscrofa10.2) using Tophat 2.0.12 with default parameters which included: enable use of GTF file, set minimum anchor length of 8, accept zero mismatches in the anchor region, allow intron length between 50 and 500,000, and allow up to 20 alignments to the reference for a given read. For annotation of genes, we used the GTF file for Sscrofa10.2 from Ensembl version 71.
The number of reads uniquely mapped to each gene was determined using Htseq-count (v0.5.3.p3). Reads that were assigned to more than one gene were not counted by Htseq-count.
The read counts per gene were normalised to counts per million (CPM). Genes expressed at very low levels were removed by keeping only those genes that achieve CPM above four in at least half the samples. Trimmed mean of M-values (TMM) normalisation was applied to this dataset to account for compositional differences between the libraries. Genes that respond to PRRSV infection at any of the day after infection compared to the day 0 baseline were determined in edgeR using a generalized linear model that takes into account the following: dpi, gender, batch (separate experiment on PHGC3 and PHGC5 animals), post globin depletion RIN score, population structure (three PCAs based on genotype of 44 animals).
Genome_build: Sus scrofa 10.2
Supplementary_files_format_and_content: Tab-delimited text files include count values for each sample.
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| Submission date |
Feb 29, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Payam Vahmani |
| E-mail(s) |
[email protected]
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| Organization name |
University of California Davis
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| Department |
Department of Animal Science
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| Street address |
One Shields Avenue
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| City |
Davis |
| State/province |
California |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platform ID |
GPL11429 |
| Series (1) |
| GSE78762 |
Genetic architecture of gene expression underlying variation in host response to porcine reproductive and respiratory syndrome virus infection |
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| Relations |
| BioSample |
SAMN04521412 |
| SRA |
SRX1605317 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2075890_GCswine-5072-7DPI-WB-7454GD-mRNA.stats.txt.gz |
100.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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