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Sample GSM1385428 Query DataSets for GSM1385428
Status Public on Jul 14, 2014
Title WT-ALUM-KP
Sample type SRA
 
Source name Lung
Organism Mus musculus
Characteristics strain: BALB/c
genotype: wild type
intraperitoneal sensitization: Alum
aerosol exposure: None
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 WTalum6_vs_WTova6_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
Lab Stokes Peebles, PI
Street address D-7235 Medical Center North, 1161 21st Avenue South
City Nashville
State/province TN
ZIP/Postal code 37232-2581
Country USA
 
Platform ID GPL17021
Series (1)
GSE57602 RNAseq analysis of mouse lung transcriptome from allergic and non-allergic mice prior to and following lung Klebsiella pneumoniae infection
Relations
BioSample SAMN02776928
SRA SRX541011

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

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