GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3068334

Query DataSets for GSM3068334

Status

Public on Sep 01, 2018

Title

WT 3

Sample type

SRA

Source name

Lungs

Organism

Mus musculus

Characteristics

strain: C57BL/6
genotype: Fth+/+
tissue: Lungs
infection: Mtb
time: 9 weeks post-infection

Treatment protocol

Mtb infected of Fth+/+ and Fth-/- mice

Extracted molecule

total RNA

Extraction protocol

Lungs were removed from Mtb infected mice at 9 weeks post infection, Stored in RNA later at -80. RNA was iolated using RNeasy Plus Mini Kit (Cat No./ID: 74134) as per manufacturing recommendations. We was used 200 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

All Samples genes FPKM and normalized counts values.xlsx
AS4

Data processing

Sequencing of mRNA was performed on the Illumina HiSeq2500. Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer followed by 2 rounds of poly A+ selection and conversion to cDNA. A TruSeq library generation kit was used as per the manufacturer’s instructions (Illumina, San Diego, CA). Library construction consisted of random fragmentation of the polyA mRNA, followed by cDNA production using random primers. The ends of the cDNA were repaired, A-tailed and adaptors ligated for indexing (up to 12 different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using qPCR in a Roche Light Cycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725K-825K clusters/mm2. Cluster density and quality were determined during the run after the first base addition parameters were assessed. Performed paired end 2 x 50 bp sequencing runs to align the cDNA sequences to the reference genome.
TopHat was used to align the raw RNA-Seq fastq reads to the mouse mm10 genome using the short-read aligner Bowtie (1, 2). TopHat also analyzes the mapping results to identify splice junctions between exons. Cufflinks uses the aligned reads from TopHat to assemble transcripts, estimate their abundances and test for differential expression and regulation (3). Cuffmerge merges the assembled transcripts to a reference annotation and is capable of tracking Cufflinks transcripts across multiple experiments. Finally, Cuffdiff finds significant changes in transcript expression, splicing and promoter use. Genes that meet desired criteria (i.e. fold change ≥ ± 2.0 and q value < 0.05) were further analyzed using Ingenuity’s Pathway Analysis tool.
References: 1) Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562-78.; 2) Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq.Bioinformatics. 2009;25(9):1105-11; 3) Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511-5
Genome_build: mm10
Supplementary_files_format_and_content: RPKM and normalized values for each sample

Submission date

Mar 27, 2018

Last update date

Sep 01, 2018

Contact name

Adrie JC Steyn

E-mail(s)

asteyn@uab.edu

Phone

205-996-4805

Organization name

University of Alabama at Birmingham