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| Status |
Public on May 08, 2019 |
| Title |
Listeria_Control_whole blood [MS095] |
| Sample type |
SRA |
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| Source name |
Listeria_Control
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| Organism |
Mus musculus |
| Characteristics |
mouse model: Listeria monocytogenes
condition: Control
infection protocol: Listeria monocytogenes was originally obtained from Drs. H. Rogers, K. Murphy, and E. Unanue, DNAX Research Institute, USA. Bacteria were grown in BHI broth to mid-log phase as determined by OD560 and cryopreserved in 20% glycerol/PBS at -80°C. Female C57BL/6 mice were infected by intravenous injection of 5x103 CFU of Listeria monocytogenes derived from cryopreserved stocks diluted in 200 ml of PBS. Control uninfected mice received PBS only. Whole blood samples were collected from individual infected and control treated mice on day 3 post infection.
tissue: Whole blood
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| Extracted molecule |
total RNA |
| Extraction protocol |
Blood was collected in Tempus reagent (Life Technologies) at 1:2 ratio. Total RNA was extracted using the PerfectPure RNA Blood Kit (5 PRIME). Globin RNA was depleted from total RNA (1.5–2 µg) using the Mouse GLOBINclear kit (Ambion).
Globin-reduced RNA (200 ng) was used to prepare cDNA libraries using the TruSeq Stranded mRNA HT Library Preparation Kit (Illumina). Quality and integrity of the tagged libraries were initially assessed with the HT DNA HiSens Reagent kit (Perkin Elmer) using a LabChip GX bioanalyser (Caliper Life Sciences/Perkin Elmer). Tagged libraries were then sized and quantitated in duplicate (Agilent TapeStation system) using D1000 ScreenTape and reagents (Agilent). Libraries were normalised, pooled and then clustered using the HiSeq® 3000/4000 PE Cluster Kit (Illumina). The libraries were imaged and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq® 3000/4000 SBS kit (Illumina) at a minimum of 25 million paired-end reads (75 bp/100 bp) per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
MS0095_S28_L005
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| Data processing |
Raw paired-end RNA-seq data was subjected to quality control using FastQC (Babraham Bioinformatics) and MultiQC. Trimmomatic v0.36 was used to remove the adapters and filter raw reads below 36 bases long, and leading and trailing bases below quality 25. The filtered reads were aligned to the Mus musculus genome Ensembl GRCm38 (release 86) using HISAT2 v2.0.4 with default settings and RF rna-strandedness, including unpaired reads, resulting from Trimmomatic, using option -U. The mapped and aligned reads were quantified to obtain the gene-level counts using HtSeq v0.6.1 with default settings and reverse strandedness. Raw counts were processed using the bioconductor package DESeq2 v1.12.4 in R v3.3.1, and normalised using the DESeq method to remove the library-specific artefacts. Variance stabilizing transformation was applied to obtain normalised log2 gene expression values. Further quality control was performed using principal component analysis, boxplots, histograms and density plots.
Raw_counts_Blood_4_module_testing.xlsx: Raw counts generated from paired-end RNA-seq fastq files, aligned using HISAT2 and quantified using HtSeq
DESeq_normalised_counts_Blood_4_module_testing.xlsx: Normalised counts generated using the DESeq method to remove the library-specific artefacts
DESeq_VSTlog2_normalised_values_Blood_4_module_testing.xlsx: Log2 expression values generated from normalised counts using variance stabilizing transformation in DESeq2
Genome_build: Mus musculus genome Ensembl GRCm38 (release 86)
Supplementary_files_format_and_content: Excel files containing raw counts, DESeq2 normalized counts, and DESeq2 log2 expression values
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| Submission date |
Sep 12, 2018 |
| Last update date |
May 08, 2019 |
| Contact name |
Akul Singhania |
| E-mail(s) |
akul.singhania@crick.ac.uk
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| Phone |
+442037963319
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| Organization name |
The Francis Crick Institute
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| Street address |
1 Midland Road
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| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE119850 |
Global transcriptional profiling unveils the interferon network in blood and tissues across different diseases [RNA-seq_blood4_module_testing] |
| GSE119856 |
Global transcriptional profiling unveils the interferon network in blood and tissues across different diseases |
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| Relations |
| BioSample |
SAMN10038109 |
| SRA |
SRX4672746 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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