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Sample GSM5725114 Query DataSets for GSM5725114
Status Public on Dec 09, 2021
Title Activated PTBP1 cKO RNAseq 3
Sample type SRA
 
Source name Activated CD8 T cells
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
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.
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
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
 
Submission date Dec 08, 2021
Last update date Dec 10, 2021
Contact name Louise Matheson
E-mail(s) [email protected]
Organization name The Babraham Institute
Department Laboratory of Lymphocyte Signalling and Development
Street address Babraham Research Campus
City Cambridge
ZIP/Postal code CB22 3AT
Country United Kingdom
 
Platform ID GPL17021
Series (1)
GSE190512 Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells
Relations
BioSample SAMN23798225
SRA SRX13359008

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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