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| Status |
Public on Sep 30, 2015 |
| Title |
1024630 |
| Sample type |
SRA |
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| Source name |
Abomasal lymph node
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| Organism |
Ovis aries |
| Characteristics |
phenotype: Low_FEC
day post infection: 7
tissue: abomasal lymph node
pathogen: Teladorsagia circumcincta larvae (L3)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Abomasal lymph node (ALN) tissue, recovered at slaughter, was immediately cut into pieces approximately 0.5 cm3 and submerged in 10 volumes of RNAlater® (Ambion). This was stored overnight at 4 °C followed by long-term storage at -80 °C. Total RNA was extracted from the ALN tissue using Sigma TRI Reagent® (Sigma Aldrich, UK) according to the manufacturer’s instructions. Small RNAs (<200 nucleotides) and residual genomic DNA were removed with the RNeasy Mini Kit (Qiagen, Germany) and an in-solution DNase digestion (RNase-free DNase set; Qiagen, Germany) according to the manufacturer’s instructions. RNA quality was assessed using an Agilent® RNA 6000 Nano Assay on the 2100 Bioanalyzer, and total RNA was quantified using the NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, UK).
Illumina TruSeq™ libraries were prepared following the TruSeq™RNA sample preparation v2 guide (Part #15026495 Rev. B) for total RNA with the following modifications: (i) the number of PCR cycles was reduced to 10 to minimise overcycling and (2) to avoid bead contamination the PCR products were purified using a Qiagen MinElute column rather than AMPure XP beads. Libraries were visualised using an Agilent® DNA 1000 assay on the 2100 Bioanalyzer, and quantified using the Qubit® dsDNA BR assay (Invitrogen, UK) according to the manufacturer’s instructions. The indexed cDNA libraries containing the specific Illumina TruSeq adapters were sent to GATC Biotech (Kontanz, Germany), where they were pooled. Each pool was sequenced on two lanes of an Illumina HiSeq2000 with 50 bp paired-end reads.
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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 |
Illumina CASAVA 1.8.0 software used for basecalling.
Trim Galore (v0.3.3) was applied to the RNAseq reads using the default settings for paired end data. Base calls with a Phred score < 20 were removed.
Trimmed reads were mapped to the ovine genome (OARv3.1) using STAR, with the Ensembl (Ensembl 74) Ovis aries transcriptome annotation supplied. Alignments were output only if the ratio of mismatches to mapped length per read pair was less than 0.02. Only uniquely mapped reads were kept for read counts.
The mapped reads were used to estimate raw counts per gene using HTSeq (v0.5.3p3) with the union overlap resolution mode.
The Bioconductor package EdgeR (v 3.0.8) was used with R software (v 3.0.2) to analyse differential expression of read counts. Comparisons were made between HighFEC and LowFEC animals at either 7 or 14 dpi or within phenotype over time between 7 and 14 dpi. Low expression tags were filtered, keeping only genes that achieved at least one count per million (CPM) in at least 5 samples. Trimmed mean of M-values (TMM) normalisation was used to account for differences in RNA composition between samples. Data were analysed using both common and tagwise dispersions. To account for multiple testing, genes were filtered using a Benjamini and Hochberg false discovery rate (FDR) of ≤0.1 or ≤0.05 for tagwise and common dispersion analyses respectively. All genes identified as differentially expressed (DE) using common dispersion estimates were included in pathway analysis.
Pre-calculated 1-to-1 orthologs were obtained from Human version (74) using Ensembl's Biomart tool. Ingenuity® Systems Pathway Analysis (IPA; Ingenuity Systems, Redwood City, CA, USA; v18841524) was used to identify the top networks, canonical pathways, diseases and functions from DE genes
Genome_build: OARv3.1
Supplementary_files_format_and_content: Tab deliminated text files containing raw counts per gene from HTSeq
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| Submission date |
Nov 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Kathryn McRae |
| Organization name |
AgResearch
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| Department |
Animal Productivity
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| Lab |
Animal Genomics
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| Street address |
Puddle Alley
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| City |
Mosgiel |
| ZIP/Postal code |
9053 |
| Country |
New Zealand |
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| Platform ID |
GPL15670 |
| Series (1) |
| GSE63547 |
Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance |
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| Relations |
| BioSample |
SAMN03216613 |
| SRA |
SRX765376 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1551985_s3_LFd7.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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