2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
Dementia Research Institute at KCL, London, UK The Francis Crick Institute, London, UK National Institute of Chemistry, Ljubljana, Slovenia
INSDC center name
public
INSDC status
ERS26957596
SRA accession
E-MTAB-15648:Day1-activated_R1_post-24hr-pulse_0hr
Submitter Id
ArrayExpress
broker name
CD8-Positive T-Lymphocytes
cell type
not collected
collection date
human
common name
Protocols: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors through Cambridge Bioscience, NHS Blood and Transplant (UK). Ethical approval and written informed consent were obtained in accordance with the Declaration of Helsinki (2013). PBMCs were isolated using Ficoll-Paque PLUS density gradient centrifugation. Human CD8⁺ T cells were purified from PBMCs using MACS Miltenyi kits (total CD8⁺ T cells: 130-096-495; MicroBeads: 130-045-201; FcR blocking reagent: 130-059-901) according to the manufacturer’s instructions. Human CD8⁺ T cells were cultured in RPMI-1640 medium (Gibco, 52400-025) supplemented with 10% FBS, 1% penicillin–streptomycin, and 30 U/ml IL-2 (Roche). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂. Human CD8+ cells were either non-activated or activated with αCD3/αCD28 beads (1:1 bead-to-cell ratio, Gibco 11132D). During cell culture, CD8+ cells were labelled with 100 µM 4-thiouridine (4SU; Sigma T4509), replenished every 4 h for 24 h. After labelling, CD8+ cells were washed and chased with 100 mM uridine (Sigma U3750) for 0, 0.5, 1, 3, 6, or 16 hours. Day 1 and day 5 activated CD8⁺ T cells were processed in parallel, with beads removed at day 4 for the latter. At each chase time point, 1-2 × 10⁶ cells were collected for RNA extraction. Total RNA was extracted from human CD8⁺ T cells after each labelling and chase time point using TRIzol reagent, followed by column-based purification. RNA integrity was assessed by Bioanalyzer (Agilent). SLAM-Seq libraries were prepared following established protocols (Herzog et al., Nat Methods 2017), with minor modifications. Briefly, thiol-modified RNA was alkylated to introduce T>C mutations detectable in sequencing. Libraries were prepared from extracted total RNA using the Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD, V2 (Cat. No. 191.96, Lexogen GmbH), which generates forward-stranded Illumina-compatible libraries of sequences close to the 3’ end of polyadenylated RNA. The protocol involves oligo(dT)-primed reverse transcription from the 3′ poly(A) tail of mRNAs, followed by RNA degradation and synthesis of the second strand using random primers. The second strand contains Illumina-compatible linker sequences, ensuring strand specificity. After PCR amplification and purification, the resulting cDNA libraries were quantified and quality-checked by Bioanalyzer before sequencing.
description
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
1
replicate
Homo sapiens
scientific_name
Day1-activated_R1_post-24hr-pulse_0hr
title