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Sample GSM1833963

Query DataSets for GSM1833963

Status

Public on Sep 16, 2016

Title

Day 5, Animal 75

Sample type

SRA

Source name

MP-12 Vaccinated Cattle

Organism

Bos taurus

Characteristics

individual: Animal 75
cell type: circulating lymphocytes
vaccine: Rift Valley fever, MP-12 vaccine
vaccine administration: subcutaneous
time: Day 5
serologic response status: vaccinated, not protected

Treatment protocol

All cattle were injected subcutaneously or intramuscularly with the Rift Valley fever MP-12 vaccine strain.

Growth protocol

There was no growth protocol, however whole blood was collected from cattle at DAY 0–7, 10, 14 and 21 and immediately mixed at a 3:5 (blood:buffer) ratio with RNALater (Invitrogen). Samples were stored at –20°C until processing.

Extracted molecule

total RNA

Extraction protocol

Whole blood was collected at DAY 0–7, 10, 14 and 21 and immediately mixed at a 3:5 (blood:buffer) ratio with RNALater (Invitrogen). Samples were stored at –20°C until processing. To purify RNA, frozen samples were thawed at 37°C, centrifuged at 2000xG, and supernatant discarded. Remaining cell pellets were subjected to two rounds of treatment with Red Blood Cell Lysing Buffer per manufacturer’s instructions (Sigma). RNA from resulting cell pellet was initially extracted by Trizol (Invitrogen) followed by treatment with RNeasy Kit with on-column DNase digestion (Qiagen). Purified RNA was quantified by spectrophotometry on a NanoDrop (Thermo, USA) and by electrophoresis with a BioAnalyzer 2100 (Agilent, USA). Only samples with an RNA Integrity Number (RIN) greater than 8.0 were used for RNASeq analysis.
All RNASeq sequencing was performed at the Texas A&M Genetics and Bioinformatics Center following manufacturer’s instructions. cDNA was generated from RNA samples of sufficient quantity and quality with Ovation RNA-Seq System (NuGen, USA), sheared by focused ultrasound (Covartis, USA) and made into barcoded (multiplexed) libraries with Encore Rapid Library System (NuGen, USA). Samples were quantified, diluted, pooled re-analyzed with a Bioanalyzer and loaded onto a Genome Analyzer II High-throughput sequencer (Illumina, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Description

demultiplex
Sample name: 75-5_2_exp_day5

Data processing

Illumina Casava1.7 software used for basecalling.
splitBC was used to split raw barcoded data to create separate fastq files for each sample as listed in sample section
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using trimmomatic.
Reads mapped to bovine refGenome UMD3.1 whole genome using bowtie2 v2.2.5 with parameters –end-to-end -D 15 -R 2 -N 0 -L 22 -iS,1,1.15
read alignment counts to transcripts by BedTools Multicov with bovine genomic intervals provided by Illumina's Igenome annotation from NCBI
Count table (count to transcript/gene mapping) created by Seralogix's NextGen Sequencing pipeline to form one matrix file genes in rows and samples in column by sample name declared above.
Differential analysis completed by DESeq2
Genome_build: UMD3.1 bovine
Supplementary_files_format_and_content: MP-12vaccinatedCountTableMatrix.txt is a matrix of unnormalized, unfiltered read counts. First 3 columns are 1) transcript id, 2) gene symbol, 3) gene description. The remaining columns are sample data corresponding to sample names from above.
Supplementary_files_format_and_content: MP-12normalizedAbundanceTableMatrix.txt generated by DESeq2 is a matrix of normalized and filtered abundance (read counts). First 5 columns are 1) Unique Identifier, 2) Probe_ID=transcript ID maps to file MP-12vaccinatedCountTableMatrix.txt, 3) Gene Description; 4) Genbank ID, 5) Gene Symbol. The remaining columns are the normalized data by sample name defined above in Sample descriptions.

Submission date

Jul 28, 2015

Last update date

May 15, 2019

Contact name

Leslie Garry Adams

E-mail(s)

gadams@cvm.tamu.edu

Phone

979-845-9816

Organization name

Texas A&M University