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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
Street address 4401 Penn Ave
City Pittsburgh
State/province PA
ZIP/Postal code 15224
Country USA
 
Platform ID GPL19057
Series (1)
GSE119029 STAT2 Signaling Regulates Macrophage Phenotype during Influenza and Bacterial Super-infection
Relations
BioSample SAMN09910463
SRA SRX4608916

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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