NextSeq 500 paired end sequencing; RNA-seq of in vitro-generated murine T helper 17 cells
RNA-seq of in vitro-generated murine T helper 17 cells
For T cell differentiation in vitro, spleens as well as brachial, axial, inguinal and mesenteric lymph nodes were isolated and separated into single cells. Viable lymphocytes were then separated from the remaining cells in Lympholyte-M Cell Separation medium (Cedarlane # CL5030) during a 15 min centrifugation step at 1250 g. Naive CD4+ CD62L+ T cells were obtained using Milteny’s CD4+CD62 L+ T Cell Isolation Kit II (#130-093-227) following the manufacturer’s instructions. Cells were cultured in IMDM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 200 µM 2-ME, and 5 % FBS. Th17, For conventional Th17 cell differentiation, T cells were cultured in the presence of 25 ng/ml IL-6 (PeproTech #AF-200-06), 0.4 ng/ml TGFβ (PeproTech #100-21) and 4 μg/ml anti–IFN-γ antibodies (BioXCell Clone XMG1.2) for 3 days. ER stress-generated Th17 cells were obtained by a combined treatment of 25 ng/ml IL-6, and 4 μg/ml anti–IFN-γ antibodies with 2uM CPA (cyclopiazonic acid; Sigma # C1530) for 3 days. In vitro-differentiated Th17 cells obtained from IL-17ACre Rosastop-tdRFP were used for FACS. After 3 days of culture in 24-well plates, T cells were washed once with PBS and stained with APC-coupled anti-CD4 antibodies and DAPI (Molecular Probes #D1306) as a live/dead stain. CD4+ DAPI- cells were sorted according to their FP expression levels by a BD FACSAria. Samples for RNA sequencing were stored in 40 μl RNAlater at -80°C. RNA isolation was performed using Qiagen’s RNeasy Mini Plus Kit (#74134) following the manufacturer’s instructions. The RNA Integrity Number (RIN) was determined by running the samples on a Agilent Bioanalyser RNA Pico Chip (#5067- 1513). The RNA content was determined on a NanoDrop spectrometer and ranged from 0.9 – 11.1 μg RNA per sample. Two samples per experiment were chosen for RNA sequencing based on their RIN. An input of 250 ng RNA per sample was used for the subsequent generation of undirectional RNA libraries using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (#E7530S), following the manufacturer’s instructions (Figure 3). Succeeding an rRNA depletion step performed with the NEBNext® rRNA Depletion Kit (#E6310), we fragmented the RNA for 15min at 94°C to obtain RNA fragments about 200 bp in size. After adapter ligation, we used NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) to generate barcoded samples for multiplexing. PCR library enrichment was performed for 13 cycles. The quality of RNA libraries was assessed on Agilent Bioanalyser High Sensitivity DNA Chips (#5067-4626).