biostudies-arrayexpressClaire MaMus musculusSalmonhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14579This project explores the effects of inhibiting myosin IIA using para-nitroblebbistatin (pNB) on effector cytotoxic T lymphocyte (CTL) activation. Effector CTLs were differentiated from OTI TCR transgenic mice for 7 days in vitro, then stimulated (or not stimulated) with anti-CD3 for 1 hour, treated with either pNB or DMSO control.biostudies-arrayexpressLibrary Construction - Libraries were prepared for sequencing with the TruSeq Stranded mRNA kit (Illumina).Sequencing - Paired-end 100 base-pair sequencing was performed using an Illumina NovaSeq X. All 24 libraries were sequenced on 1 lane of a 10B flowcell, at the Cancer Research UK Cambridge Institute by staff blinded to sample treatments.Growth Protocol - Effector OT-I CTLs were differentiated and cultured from murine splenocytes as follows. Naïve OT-I splenocytes were stimulated with 10 nM ovalbumin peptide, SIINFEKL (OVA) for 3 days in complete mouse T cell media: RPMI 1640 medium with 10% FBS, 50 μM β-mercaptoethanol, 10 U/ml recombinant murine IL-2, 2 mM L-Glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were pelleted and resuspended in fresh media from 3 days after stimulation, expanded in CTL media and used at day 7 after stimulation.Sample Collection - Cells were harvested by centrifugation into a cell pellet, and lysed with 350 μL RLT lysis buffer supplemented with 1% v/v β-mercaptoethanol.Nucleic Acid Extraction - Lysates were passed through a Qiashredder and frozen. RNA was extracted using the RNEasy Mini Kit, according to the manufacturer’s protocol with on-column DNA digestion with RNase-free DNase I. RNA samples from 6 biological replicates from the same litter (3 males and 3 females, aged between 11 to 14 weeks) were stimulated in 4 independent experiments. RNA extraction from all 24 cell pellets were performed on the same day. RNA quality was verified by Tapestation 4150 and quantity was determined using a Qubit Fluorometer (version 4).Sample Treatment - Flat-bottom tissue culture plates were coated with 1 μg/mL anti-CD3e (clone 145-2C11, BD Biosciences) in PBS at 37°C for 1 hour. Plates were then washed in PBS. Two million OT-I CTLs were stimulated (or not stimulated) for 1 hour on anti-CD3e coated plate, treated with either 20 μM pNB or DMSO. For unstimulated control, cells were plated without antibody coating.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Reads were aligned to the mouse genome (annotation from Ensembl GRCm39 version 108) and transcript expression was quantified using Salmon (v1.10.3) (Patro et al 2017). This was performed by the Cancer Research UK Cambridge Institute bioinformatics core. Counts table from this analysis stage is also provided.Data Transformation - Processed data file is a raw counts table as described in the \\\\"high throughput sequence alignment protocol\\\\". To adjust for biases induced by transcript length, gene level counts were corrected for average transcript length using the LengthscaledTPM option within tximport for scaling. The scaled gene level counts were filtered using an internal function of edgeR (which retained genes that have a CPM of 0.24 or more in at least 6 samples for this dataset) and normalized by the method of trimmed mean of M-values (TMM). Differential gene analysis was performed following a published workflow (Law et al 2016) using edgeR (v4.2.1) (Robinson et al 2010, McCarthy et al 2012, Chen et al 2016) and limma (v3.60.4) (Ritchie et al 2015, Phipson et al 2016, Law et al 2014 ).UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNAMus musculusClaire MafalseRNA-seq of murine cytotoxic T lymphocytes in response to myosin IIA inhibition using para-nitroblebbistatinThis project explores the effects of inhibiting myosin IIA using para-nitroblebbistatin (pNB) on effector cytotoxic T lymphocyte (CTL) activation. Effector CTLs were differentiated from OTI TCR transgenic mice for 7 days in vitro, then stimulated (or not stimulated) with anti-CD3 for 1 hour, treated with either pNB or DMSO control.2025-09-27T00:00:00Z2025-09-27T01:04:09.494Z2024-11-03T22:56:10.407ZE-MTAB-14579ERP165832EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969