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Sample GSM5725109

Query DataSets for GSM5725109

Status

Public on Dec 09, 2021

Title

Activated Ctrl RNAseq 2

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: Ptbp1-fl/fl
Sex: Female
age: 13 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