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Sample GSM1385431

Query DataSets for GSM1385431

Status

Public on Jul 14, 2014

Title

STAT6KO-OVA-KP

Sample type

SRA

Source name

Lung

Organism

Mus musculus

Characteristics

strain: BALB/c
genotype: STAT6KO
intraperitoneal sensitization: OVA-Alum
aerosol exposure: OVA
infection: Klebsiella pneumoniae

Extracted molecule

total RNA

Extraction protocol

For each mouse, the right lung was removed, placed in a sterile eppendorf tube, and snap frozen in liquid nitrogen. RNA extraction was performed on each sample by TRIZOL extraction following by Qiagen RNEasy Mini kit purification and on-column DNAse digestion.
The Vanderbilt Technologies for Advanced Genomics Core Facility (VANTAGE, Nashville, TN) used the Illumina TruSeq Stranded mRNA Sample Preparation Kit to convert the mRNA in 100 ng of total RNA into a library of template molecules suitable for subsequent cluster generation and sequencing on the Illumina HiSeq 2500. The first step was to do a quality check of the input total RNA by running an aliquot on the Agilent Bioanalyzer to confirm integrity. The Qubit RNA fluorometry assay was used to measure concentration. The input to library prep was 50 ul of 100 ng of total RNA (2 ng/ul). The total RNA underwent enrichment of the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following enrichment, the eluted poly(A) RNA was cleaved into small fragments of 120-210 bp using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase and random hexamer primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments then went through an end repair process, the addition of a single ‘A’ base, and then ligation of the Illumina multiplexing adapters. The products were then purified and enriched with PCR to create the final cDNA sequencing library. The cDNA library underwent quality control by running on the Agilent Bioanalyzer HS DNA assay to confirm the final library size and on the Agilent Mx3005P qPCR machine using the KAPA Illumina library quantification kit to determine concentration. A 2 nM stock was created and samples pooled by molarity for multiplexing. From the pool 12 pM was loaded into each well for the flow cell on the Illumina cBot for cluster generation. The flow cell was then loaded onto the Illumina HiSeq 2500 for Paired-end 50 bp sequencing utilizing v3 chemistry and HTA 1.8.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

KOalum6_vs_KOova6_gene_exp_significant.diff

Data processing

TopHat 2.0.9, for reads alignment, command line with parameter is, tophat2 --segment-length 25 -r 123 -G /path_to_annotation/mm10/Mus_musculus.GRCm38.68.gtf -p 6 -o tophat_output /path_to_reference/mm10/bowtie2_index/mm10 sample_1.fastq sample_2.fastq
Cufflinks 2.0.2/cufflinks, command line with parameter is, cufflinks -p 6 -o cufflink_output path_to_each_sample_aligned.bam
Cufflinks 2.0.2/cuffmerge, command line with parameter is, cuffmerge -o merged_gtf_output -p 6 -s /path_to_reference/mm10/mm10.fa -g /path_to_annotation/Mus_musculus.GRCm38.68.gtf sample_gtf_file_list.txt
Cufflinks 2.0.2/cuffdiff, for group comparasion by FPKM value, command line with parameter is, cuffdiff -o output_folder -p 6 -L group_1,group_2 -u -b /path_to/reference/mm10/mm10.fa merged.gtf
filter for sigmificant gene expression from cuffdiff gene_exp.diff file with q_value <=0.01 and log2(fold_change) >=1.5.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files from Cuffdiff output including the gene_exp.diff for the FPKM based comparasions of group KOalum0hour_vs_KOova0hour, WTalum0hour_vs_WTova0hour KOalum6hour_vs_KOova6hour, and WTalum6hour_vs_WTova6hour.

Submission date

May 13, 2014

Last update date

May 15, 2019

Contact name

Daniel E Dulek

E-mail(s)

daniel.dulek@vanderbilt.edu

Organization name

Vanderbilt University Medical Center

Department

Pediatrics