biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsInés Pineda-TorraNextSeq 500RNA-seq of total RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-9141We used RNA-sequencing to determine the transcriptional response to liver X receptor (LXR) activation in primary human CD4+ T cells, both at rest and under activation conditions. CD4+ T cells were isolated from 3 healthy donors and treated with the synthetic LXR agonist GW3965 (GW) for 24 hours. Gene expression was compared to control samples (CTRL) treated with the LXR antagonist GSK1440233. To activate the T cell antigen receptor (TCR), cells were pre-treated with CTRL/GW alone for 6 hours then transferred to plates containing anti-CD3 and anti-CD28 for a further 18 hours.biostudies-arrayexpressSequencing - Libraries to be multiplexed in the same run are pooled in equimolar quantities, calculated from Qubit and Bioanalyser fragment analysis. Samples were sequenced on the NextSeq 500 instrument (Illumina, San Diego, US) using a 43bp paired end run. Run data were demultiplexed and converted to fastq files using Illumina’s bcl2fastq Conversion Software v2.19.Library Construction - Samples were processed using the NEB RNA Ultra II Directional assay with PolyA mRNA workflow (p/n E7760) according to manufacturer’s instructions. Briefly, mRNA was isolated from 100ng total RNA using Oligo dT beads to pull down poly-adenylated transcripts. The purified mRNA was fragmented using chemical hydrolysis (heat and divalent metal cation) and primed with random hexamers. Strand-specific first strand cDNA was generated and “A-tailed” at the 3’ end. Full length xGen adaptors (IDT), containing unique 8bp dual sample specific indexes, a unique molecular identifier and a T overhang are ligated to the A-Tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle PCR (13 cycles).Sample Collection - Peripheral blood mononuclear cells (PBMCs) were isolated from three leukocyte cones purchased from the NHS Blood and Transplant Service using Ficoll-Paque PLUS [GE Healthcare]. PBMCs were cryopreserved in heat inactivated foetal bovine serum [Labtech] + 10% dimethylsulfoxide [Sigma, Cat# D2650] until use. Upon thawing, CD4+ T cells were negatively isolated using magnetic assisted cell sorting (MACS) [EasySep Human CD4+ Enrichment Kit, Stem Cell, #19052].Nucleic Acid Extraction - Total RNA was extracted using TRIzol reagent (Life technologies) as per the manufactrer's instructions. DNA was removed with the DNA-free™ DNA Removal Kit [Invitrogen, #AM1906].Sample Treatment - Purified T cells were cultured in in 96 well plates (1 x106 cells/well) in RPMI 1640 media [Sigma] supplemented with 10% heat-inactivated foetal bovine serum [Labtech] and 20 ug/mL gentamycin [Sigma]. T cells (3 x 106) were treated with the LXR antagonist GSK1440233 (CTRL, 1 µM) [Sigma] or the LXR agonist GW3965 (GW, 2 µM) [Sigma, CAS: 405911-17-3] for 24 hours. To activate the T cell antigen receptor (TCR) cells were stimulated with 1 µg/mL plate bound anti-CD3 [clone UCHT1, Thermo Fisher Scientific Cat# 16-0038-85, RRID:AB_468857] and 1 µg/mL soluble anti-CD28 [clone CD28.2, Thermo Fisher Scientific Cat# 16-0289-81, RRID:AB_468926] for the last 18 hours of the culture.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesInés Pineda-TorraKirsty WaddingtonfalseRNA-seq of primary human CD4+ T cells treated with the LXR agonist GW3965, with and without TCR stimulationWe used RNA-sequencing to determine the transcriptional response to liver X receptor (LXR) activation in primary human CD4+ T cells, both at rest and under activation conditions. CD4+ T cells were isolated from 3 healthy donors and treated with the synthetic LXR agonist GW3965 (GW) for 24 hours. Gene expression was compared to control samples (CTRL) treated with the LXR antagonist GSK1440233. To activate the T cell antigen receptor (TCR), cells were pre-treated with CTRL/GW alone for 6 hours then transferred to plates containing anti-CD3 and anti-CD28 for a further 18 hours.2021-04-30T00:00:00Z2022-02-04T04:58:54.017Z2022-02-04T04:58:54.017ZE-MTAB-9141ERP122047EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0003969EFO_0004184