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Sample GSM3193171

Query DataSets for GSM3193171

Status

Public on Dec 20, 2018

Title

IFNPR8CC-3_S12

Sample type

SRA

Source name

IFNAR2-/- lung lobes, PR8 Influenza virus, 9h post intranasal

Organism

Mus musculus

Characteristics

tissue: LUNG LOBES
mouse strain: IFNAR2-/-
infection: PR8, 9h post infection (intranasal)

Extracted molecule

total RNA

Extraction protocol

Lung lobes were processed as described in Ma et al. ~3000 cells were collected for each sample. Total RNA was extracted from sorted cells using the RNeasy Micro Kit (Qiagen).
First strand cDNA synthesis and tailing by reverse transcription was performed using Clontech’s proprietary SMART (Switching Mechanism at 5’ End of RNA Template) technology. Following first strand synthesis, cDNA was amplified 13 cycles by LD PCR using blocked PCR primers. Amplified cDNA was purified using AMPure XP prior to QC using the Bioanalyser 2100 HS DNA kit (Agilent Technologies). Library preparation of purified amplified cDNA was performed following Nextera XT library preparation (Illumina). Following QC, 150pg of cDNA was tagmented (simultaneously fragmented with adaptors inserted) using Nextera transposon. Molecular barcodes were incorporated during 12 cycles of amplification followed by purification using AMPure XP. The libraries passed a quality checkpoint (Qubit and Bioanalyser HS DNA) prior to normalisation and pooling before loading onto a HiSeq 2500 (Illumina) sequencer for paired-end sequencing. The libraries were each sequenced to 10-20 million reads.
RNA-seq and library construction were performed as 4 independent batches.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Batch 3 - 151102_SN7001291_0303_BH5N3GADXX
counts.tsv

Data processing

RNA-seq reads were aligned to the mouse genome mm10 using STAR (Dobin et al., 2013). Aligned fragments were summarised by gene (mm10 annotation from Ensembl) using featureCounts (Liao et al., 2014), ignoring those where both reads of the pair had mapping quality scores <10. Raw counts were transformed to log Counts Per Million (CPM) for visualisation. Normalisation and differential expression testing was conducted using edgeR (Robinson et al., 2010). The HTSFilter package (Rau et al., 2013) was used to remove genes with consistent low expression following normalisation and testing as suggested by the HTSFilter documentation.
For the IAV genome, RNAseq reads were aligned to a synthetic genome containing the 8 gene segments of the PR8 virus retrieved from NCBI using Tophat (Trapnell et al., 2009). Aligned reads were assigned to the appropriate viral segments using feature Counts function from Bioconductor Rsubread package (Liao et al., 2013). Differential expression analysis was performed on normalised counts in every viral segment by the total (virus and mouse) number of reads in each sample with limma package (Ritchie et al., 2015), treating every viral segment as a gene.
Genome_build: mouse genome mm10

Submission date

Jun 17, 2018

Last update date

Dec 20, 2018

Contact name

Joel Zhi-Iong Ma

E-mail(s)

intuitivelogic.au@gmail.com

Organization name

University of Melbourne

Street address

Grattan Street