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| Status |
Public on Apr 08, 2014 |
| Title |
ILTV_RNASeq_Inoculated_1 |
| Sample type |
SRA |
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| Source name |
Tracheal scraps
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| Organism |
Gallus gallus |
| Characteristics |
sample collection time: Six days post inoculation (6 dpi)
breed: Leghorn
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| Treatment protocol |
Ten, 15-day-old SPF white leghorn chickens were obtained (Charles River, Wilmington, Massachusetts, USA) and divided into two groups of 5 birds each. At 21 days of age, one group of 5 birds was inoculated with sterile vaccine diluent without addition of the lyophilized Trachivax® vaccine (50 μL per nostril, 50 μL per eye; 200μL total) and placed in a separate animal room as a control group. The vaccine was then prepared according to the manufacturer's recommendation for the eye drop administration method. The second group of birds was then inoculated with Trachivax® vaccine (50 μL per nostril, 50 μL per eye; 200μL total at a concentration of 3.3 x 103 pfu) and were housed as a group in an animal isolator. All animal experiments and procedures were performed under Institutional Animal Care and Use Committee (IACUC) guidelines and approval
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| Extracted molecule |
total RNA |
| Extraction protocol |
mRNA was extracted directly from tracheal samples using the Oligotex Direct mRNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. First strand cDNA was generated using the SuperScript II Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) with random hexamers (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions in a final volume of 40µl. Second strand cDNA was synthesized using DNA polymerase I (NEB, Ipswich, MA, USA) supplemented with 30nmol dNTPs and 2U RNase H (Invitrogen, Carlsbad, CA, USA), incubated at 25° C for 2.5 hours, then purified with QIAquick PCR purification kit (Qiagen, Valencia, CA, USA).
Libraries were constructed using Multiplexing Sample Preparation Oligonucleotide Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. Briefly, the double stranded cDNA (dscDNA) was sonicated to approximately 200-500bp using a Bioruptor sonicator (Diagenode, Denville, NJ, USA) in a final volume of 50µl for 40 minutes. The resulting fragments were repaired using End repair module (NEB, Ipswich, MA, USA) and 3′A was added by Klenow Fragment (NEB, Ipswich, MA, USA). The Solexa adaptors (Illumina, San Diego, CA, USA) were then ligated to the dscDNA fragments by T4 DNA ligase (NEB, Ipswich, MA, USA). PCR with 18 cycles was performed using the adaptor primers and index primers. PCR products with a length of 200-500bp were isolated by QIAquick Gel purification kit (Qiagen, Valencia, CA, USA). Clustering and sequencing then was performed on the Hi-Seq 2000 (Illumina, San Diego, CA, USA) following the manufacturers protocols.
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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 |
Sequencing using Illumina HiSeq 2000
Alignment to the galGal3 genome reference were obtained with Bowtie, trimming the first 15 5' nucleotides, resulting in reads of 35 bases
Aligned bam files were transformed to bed files, using the bamtobed function in bedtools
Normalized reads counts was obtained using GenomicRanges and DESeq
The final list of genes with their respective fold change, log2 fold change , p-value and adjusted p value was obtained using DESeq
Genome_build: Gallus_gallus-2.1 (galGal3)
Supplementary_files_format_and_content: Count_Normalized.txt is a matrix where each row represents a gene. The first column shows gene names, the other columns are normalized counts for each sample. Columns labeled KZ59-10 and KZ59-11 correspond to inoculated, and KZ59-9 to control
Supplementary_files_format_and_content: GenesTable.txt is a matrix with columns for gene id, baseMean across samples, baseMeanA corresponds to inoculated and baseMeanB to control, fold changes between conditions, log2fold change between conditions, the p-value and the adjusted p-value for each gene.
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| Submission date |
Apr 15, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jose Adrian Carrillo |
| E-mail(s) |
carrillo@umd.edu
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| Organization name |
University of Maryland
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| Department |
Department of Animal and Avian Sciences
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| Lab |
Song's lab
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| Street address |
1413 Animal Sciences Center
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| City |
College Park |
| State/province |
MD |
| ZIP/Postal code |
20742-2311 |
| Country |
USA |
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| Platform ID |
GPL16133 |
| Series (1) |
| GSE46046 |
Transcriptome analysis reveals an activation of MHC-I and MHC-II pathways in chicken trachea immunized with infectious laryngotracheitis virus vaccine |
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| Relations |
| SRA |
SRX265302 |
| BioSample |
SAMN02045694 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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