|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Dec 20, 2018 |
Title |
IFNPR8AECII-3_S11 |
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
|
City |
Parkville |
State/province |
Victoria |
ZIP/Postal code |
3010 |
Country |
Australia |
|
|
Platform ID |
GPL17021 |
Series (1) |
GSE115904 |
Unique transcriptional architecture in airway epithelial cells and macrophages shapes distinct responses following influenza virus infection ex vivo. |
|
Relations |
BioSample |
SAMN09434691 |
SRA |
SRX4224437 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|