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GEO help: Mouse over screen elements for information. |
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Status |
Public on Dec 09, 2021 |
Title |
Activated PTBP1 cKO RNAseq 3 |
Sample type |
SRA |
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Source name |
Activated CD8 T cells
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 tissue: Spleen + LN treatment: 24h activation, anti-CD3 + anti-CD28 + IL2 genotype: CD4-CreTg Ptbp1-fl/fl Sex: Female age: 9 weeks
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Treatment protocol |
Naïve cells were either used unstimulated, or were activated ex vivo for 24h by culturing in RPMI 1640 Medium (Dutch modification) with 20 mM HEPES (ThermoFisher cat#:22409-015), 50 μM b-Mercaptoethanol, 100 units/ml penicillin and streptomycin, GlutaMAX and 10% FCS on 96 flat-bottom well plates precoated with 5μg/ml anti-CD3 (2C11) and 1μg/ml anti-CD28 (37.51) antibodies, in the presence of 20ng/ml murine IL2.
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Growth protocol |
Single cell suspensions were prepared from spleen and lymph nodes by passing the organs through 70 and 40 mm cell strainers consecutively. Negative naïve CD8 T cell isolation was achieved by depletion of cells bound by biotinylated antibodies: CD4 (GK1.5), CD11b (M1/70), CD11c (N418), CD19 (1D3), B220 (A3-6B3), CD105 (MJ7/18), TER-119, gamma/delta TCR (GL3), Gr1 (RB6-8C5), NK1.1 (PK136), F4/80 (BM8), CD44 (1M7) and magnetic Dynabeads™ M-280 Streptavidin (Thermofisher cat#:11206D), except for iCLIP experiments where the anti-CD44 antibody was omitted.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted from naïve and activated CD8 T cells using the RNeasy Micro Kit (cat# 74004, Qiagen). 10 ng RNA were used and 7 PCR cycles were carried out to synthesise cDNA with the SMART-Seq® v4 Ultra® Low Input RNA Kit (cat # 634891, Takara). RNAseq libraries were prepared with the Nextera XT DNA Library Preparation Kit (cat# FC-131–1096, Illumina) and carrying out 8 PCR cycles. mRNA-seq libraries were run across two lanes of a HiSeq 2500 RapidRun, generating 125bp paired end reads.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Reads were trimmed with Trim Galore (v0.6.1) to remove adapters and poor quality base calls Trimmed reads were mapped to the GRCm38 mouse genome using HISAT2 (v2.1.0), suppressing unpaired or discordant alignments and taking into account known splice sites from the Ensembl Mus_musculus.GRCm38.90 annotation. BAM files were imported into Seqmonk (v1.45.0), and raw read counts generated over mRNA features with merged isoforms, using the Ensembl Mus_musculus.GRCm38.96 annotation, assuming unstranded, paired reads. FPKMs were calculated using the same annotation, using cuffnorm from Cufflinks (v2.2.1), with geometric normalisation. Genome_build: GRCm38 Supplementary_files_format_and_content: RNAseq_raw_counts.tsv: tab-delimited text file containing gene names and IDs together with raw counts for all RNAseq samples, generated with Seqmonk. Supplementary_files_format_and_content: RNAseq_FPKM.tsv: tab-delimited text file containing gene names and IDs together with FPKM values for all RNAseq samples, generated with cuffnorm from Cufflinks. Supplementary_files_format_and_content: iCLIP processed data files: bedgraph files for each replicate containing significant xlink-binding sites (FDR<0.05) identified with iCOUNT
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Submission date |
Dec 08, 2021 |
Last update date |
Dec 10, 2021 |
Contact name |
Louise Matheson |
E-mail(s) |
[email protected]
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Organization name |
The Babraham Institute
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Department |
Laboratory of Lymphocyte Signalling and Development
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Street address |
Babraham Research Campus
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City |
Cambridge |
ZIP/Postal code |
CB22 3AT |
Country |
United Kingdom |
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Platform ID |
GPL17021 |
Series (1) |
GSE190512 |
Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells |
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Relations |
BioSample |
SAMN23798225 |
SRA |
SRX13359008 |
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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