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Sample GSM3356804

Query DataSets for GSM3356804

Status

Public on Dec 26, 2018

Title

WT 2

Sample type

SRA

Source name

mouse lung

Organism

Mus musculus

Characteristics

strain: C57BL/6Tac
genotype/variation: wild type

Treatment protocol

Mice were infected with 100 PFU of influenza A/PR/8/34 (FLU) in 50 ul of sterile PBS by oropharyngeal aspiration. Six days later mice were challenged with 5x10^7 MRSA USA300 in 50ul of sterile PBS by oropharyngeal aspiration for one additional day

Extracted molecule

total RNA

Extraction protocol

Whole lung RNA was isolated using the Agilent Absolutely RNA Miniprep Kit
Total RNA libraries were generated using Illumina TruSeq Stranded mRNA sample preparation kit. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by using dUTP in the Second Strand Marking Mix, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library.The cDNA libraries are validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality is examined using Agilent Tapestation 2200.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw sequencing reads from Illumina sequencing platform that was converted into FASTQ file format were quality checked for potential sequencing issues and contaminants using FastQC.
Adapter sequences, primers, Ns, and reads with quality score below 28 were trimmed using fastq-mcf of ea-utils and Trimmomatic. Reads with a remaining length of fewer than 20bp after trimming were discarded.
Single reads were mapped to the mouse genome (m10) using STAR in a strand specific manner.
Cufflinks was used to determine FPKM levels for each gene from the STAR alignment and was used as input for Cuffdiff.
Read counts were then normalized across all samples and significant differentially expressed genes were determined by adjusted P-value with a threshold of 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: .diff file. Contains differential gene expression results between WT vs STAT2-/-. Includes gene_ID, locus, log2 (fold_change), test_stat, p_value, and q_value.

Submission date

Aug 24, 2018

Last update date

Dec 26, 2018

Contact name

John F Alcorn

E-mail(s)

john.alcorn@chp.edu

Organization name

Children's Hospital of Pittsburgh

Department

Pediatrics