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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 01, 2019 |
| Title |
CXCR5+_Ifnar1ko_IL27_2 |
| Sample type |
SRA |
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| Source name |
CD8+ T cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
infection: LCMV clone 13
cell type: CXCR5+ CD8+ T cells
tcr specificity: undetermined
donor mice: Ifnar1-/-
flow cytometry population: CD8+ CXCR5+ B220-
treatment: IL-27, 12 h
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| Treatment protocol |
Splenic CXCR5+ CD8+ and CXCR5- CD8+ T cells were purified by flow cytometry, plated at a concentration of 1 x 10^6 cell/ml and treated with growth medium (RPMI1640 containing 10% FBS, and supplemented with Non-essential amino acids, sodium pyruvate, HEPES, L-Glutamine, 2-Mercaptoethanol, Penicillin-Streptomycin (Gibco)) with or without recombinant IL-27 (20 ng/ml, R&D Systems, Minneapolis, MN) or recombinant IFN-β (1000 U/ml, carrier-free, PBL Assay Science) for 12 h at 37°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mouse spleens were processed into splenocytes, enriched for mouse T cells using a magnetic negative selection (STEMCELL Technologies), stained and T cells were isolated by flow cytometry. RNA isolation from sorted cells was performed using the PicoPure RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA). RNA integrity was analyzed using Agilent RNA Nano Kit (Agilent, Santa Clara, CA).
Amplified cDNA was prepared using SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, Mountain View, CA), sheared using Covaris tubes (Covaris, Woburn, MA) and Illumina libraries constructed using the NEBNext Ultra DNA Library Prep Kit (NEB, Ipswich, MA) and sequenced using the NextSeq 500 system (Illumina, San Diego, CA) at a depth of 20-25 million single-end 75 bp reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
PolyA RNA
raw_counts.txt
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| Data processing |
Quality of raw RNA-sequencing reads was checked using FastQC version 0.11.3 (Barbaham Institute, Cambridge, UK; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Illumina’s bcl2fastq Conversion Software (version 2.17.1.14) was used to de-multiplex reads and convert BCL files generated by the Illumina NextSeq 500 to FASTQ.
Reads were mapped to the mouse genome (GRCm38 assembly with Ensembl v87 transcriptome annotations) and gene counts calculated using STAR version 2.5.2a (Dobin et al., 2013) using the following parameters: --quantMode GeneCounts –outSAMtype None –sjdbGTfile /Ensembl/Mus_musculus.GRCm38.87.gtf –sjdbOverhang 74
Genome_build: GRCm38
Supplementary_files_format_and_content: raw_counts.txt: Tab-delimited text file includes raw gene counts.
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| Submission date |
Sep 06, 2018 |
| Last update date |
Apr 01, 2019 |
| Contact name |
John R Teijaro |
| E-mail(s) |
teijaro@scripps.edu
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| Organization name |
The Scripps Research Institute
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| Department |
Immunology and Microbial Science
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| Street address |
10550 North Torrey Pines Rd
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE97139 |
The role of IFN-I and IL-27 in the expansion of virus-specific CD8+ T cell subtypes during persistent infection |
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| Relations |
| BioSample |
SAMN09986476 |
| SRA |
SRX4650657 |
| 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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