title: NextSeq 500 paired end sequencing; RNA-seq of primary human CD4+ T cells treated with the LXR agonist GW3965, with and without TCR stimulation
design: RNA-seq of primary human CD4+ T cells treated with the LXR agonist GW3965, with and without TCR stimulation
library_construction_protocol: 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].   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.  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].  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).