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Sample GSM5884268

Query DataSets for GSM5884268

Status

Public on Feb 10, 2022

Title

Activated_P1KO_ONT_1

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: 10 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).
ONT cDNA-PCR libraries were generated essentially according to the CELLO-seq protocol (Berrens et al, 2021, Nat. Biotech.; doi:10.1038/s41587-021-01093-1) with the following modifications: The addition of UMIs by splint ligation was omitted. Reverse transcription was performed using 1 ng total RNA isolated from a pool of cells. PCR reactions were performed using standard Oxford Nanopore Technologies barcoded primers under the following conditions: 90 °C for 45 s, 20 cycles (90 °C for 15 s, 64 °C for 30 s, 72 °C for 10 min), 72 °C for 5 min. PCR products were purified using 0.6X AMPure XP beads (Beckman). Equal quantities of each cDNA sequencing library were pooled using a total of 200 fmol, before sequencing on a MinION R9.4.1 flow cell using the SQK-LSK109 kit (1D sequencing). The standard MinKNOW procedure was used for a 72 hour run, according to manufacturers’ instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Data processing

Raw reads were basecalled using Guppy (4.0.11), in fast mode.
Sequence reads with a mean sequence quality of 7 were classified as passed reads; these were first demultiplexed with the Guppy debarcoder option and then processed with pychopper (2.5.0) in order to identify, orient and trim the full-length cDNA reads.
Pychopped reads were aligned with minimap2 (2.17) with splice aware setting (-ax splice), which is compatible with pipelines for further isoform identification analysis. The sequences were aligned to mouse genome reference GRCm38.
Aligned read counts over each gene were quantified using SeqMonk (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Reads were imported with the parameters Single End; Dedup=No; MAPQ>=20, and without treating as RNA-seq data in order to prevent splitting of spliced reads. Feature probes over genes from the GRCm38.96 annotation were then generated and reads quantified, only including those mapping to the same strand as the gene.
Genome_build: GRCm38
Supplementary_files_format_and_content: ONT_cDNAPCR_raw_counts.tsv: tab-delimited text file containing gene names and IDs together with raw counts over each gene for each long-read ONT RNA sequencing library, generated with Seqmonk.

Submission date

Feb 09, 2022

Last update date

Feb 10, 2022

Contact name

Louise Matheson

E-mail(s)

[email protected]

Organization name

The Babraham Institute

Department

Laboratory of Lymphocyte Signalling and Development