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| Status |
Public on Jul 06, 2015 |
| Title |
B6d14_3 |
| Sample type |
SRA |
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| Source name |
lungs
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| Organism |
Mus musculus |
| Characteristics |
tissue: lungs
strain: B6
infection: influenza
time: 14 d
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| Treatment protocol |
Mice were sacrificed and entire lungs were extracted from both mouse strains on days 1, 3 and 5 after infection. For mock-treated animals, mice were sacrificed at days 1 and 3 post treatment. In addition, lungs from C57BL/6J mice were also collected on days 8 and 14. For every treatment and day post infection (p.i.) 4-5 mice were prepared. The lungs were immediately transferred to RNAlater solution (Qiagen), kept at 4°C for one day and subsequently stored at -20°C. RNA was isolated using Qiagen Midi Kit as described previously (Alberts et al., 2010). RNA quality was controlled on a 2100 Bioanalyzer Instrument (Agilent). All RNA samples had a RIN of ≥9.7. Three independent biological replicates were selected for each time point for subsequent RNA sequencing.
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| Extracted molecule |
total RNA |
| Extraction protocol |
20µg of total RNA was enriched for poly A+ RNA using one cycle of the Poly A Purist Kit from Ambion according to manufacturer’s standard protocol. The resulting enriched RNA samples were then analyzed on an Agilent Bioanalyzer to determine the remaining amount of rRNA in the samples. If the amount was higher than 5%, samples were subjected to another cycle of Poly A enrichment. 100ng of the poly A+ enriched RNA was then used to prepare libraries for sequencing using the AB Library Builder™ Whole Transcriptome Core Kit for 5500 Genetic Analysis Systems on a Library Builder system. Libraries were amplified for 15 cycles before 5500 Wildfire primers were added using five cycles of fusion primer amplification as directed in the 5500 Wildfire manual. Before sequencing, small aliquots of libraries were pooled and sequenced on an Ion Torrent PGM 314 chip after additional amplification with PGM fusion primers. The library pools were quantified by Real-Time PCR and immobilized on flowcells for the SOLiD 5500 Wildfire instrument (Applied Biosystems) and sequenced (50bp reads). The average number of reads per sample was 29.5. One sample had high number of reads (230 million) and the others on average had 23 million reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500 Genetic Analyzer |
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| Data processing |
The mouse reference genome (GRCm38/mm10) was downloaded from ftp://hgdownload.cse.ucsc.edu/goldenPath/mm10/chromosomes/. Sequencing reads (XSQ format) from C57BL/6J samples were aligned to the C57BL/6J reference genome using the Whole Transcriptome mapping module of the LifeScope 2.5.1 software (http://www.lifetechnologies.com/lifescope). Similarly, sequencing reads from DBA/2J samples were aligned to the enhanced DBA/2J genome that was generated by substituting ~4.5 million DBA/2J SNPs in the reference genome. Filter reference containing polyA, polyC, polyG, polyT, rRNAs, tRNAs, as well as adaptor, barcode, and primer sequences was used to remove non-mRNAs reads prior to the mapping. We used the mouse RefSeq transcript annotation downloaded from UCSC genome browser (www.genome.ucsc.edu) to generate a junction reference library containing a list of exon-exon pairs. Reads were aligned against both the reference genome and the junction library. Reads that could not be mapped were realigned against the H1N1 viral contigs. Reads with minimum mapping quality of 10 were used to generate raw counts to be used for downstream differential and correlation analysis.
DESeq2 statistical package was used to normalize and log transform raw read counts producing ‘normalized expression counts’. The ‘normalized expression counts’ were then used for further analysis.
Genome_build: mm10
Supplementary_files_format_and_content: normalized counts
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| Submission date |
Feb 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert Geffers |
| E-mail(s) |
robert.geffers@helmholtz-hzi.de
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| Phone |
+49 531-6181-3058
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| Organization name |
HCI - Helmholtz Centre for Infection Research
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| Department |
Dep. Molecular Bacteriology
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| Lab |
Genome Analytics
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| Street address |
Inhoffenstr. 7
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| City |
Braunschweig |
| ZIP/Postal code |
38124 |
| Country |
Germany |
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| Platform ID |
GPL16790 |
| Series (1) |
| GSE66040 |
RNA-seq influenza infected B6 & D2 mice |
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| Relations |
| BioSample |
SAMN03352397 |
| SRA |
SRX882995 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1613358_B6d14_3.txt.gz |
225.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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