Illumina HiSeq 2000 paired end sequencing; Effect of the IRE1a-XBP1 pathway inhibition during T helper 2 differentiation
Effect of the IRE1a-XBP1 pathway inhibition during T helper 2 differentiation
In vitro differentiated and restimulated T helper 2 (Th2) cells (4m8c treated and untreated) were washed with PBS and the cell pellets were used for RNA extraction.  Spleens from 6-12 weeks old C57BL/6 mice were used as a source of naive T cells.  Splenic naive T helper cells were purified with the CD4+CD62L+T Cell Isolation Kit II (Miltenyi Biotec) and polarized in vitro toward differentiated Th2 subtype as described before in (Mahata et al., 2014). In brief, naive cells were seeded into anti-CD3e (2 μg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 μg/ml, clone 37.51, eBioscience) antibody coated 96-well round bottom plates. The medium contained the following cytokines and/or antibodies for Th2 subtype: recombinant murine IL-2 (10 ng/ml, R&D Systems), recombinant murine IL-4 (10 ng/ml, R&D Systems) and neutralizing anti-IFN-g (5μg/ml, cloneXMG1.2eBioscience). The cells were removed from the activation plate on day 4 (72 hours).  Th2 cells were cultured for another two days in the absence of anti-CD3 and CD28 stimulation. Then, cells were restimulated by anti-CD3e/CD28 coated plate for 6 hrs. For flow cytometric detection cells were treated with monensin (2μM, eBioscience) for the last 3 hours. Negatively selected splenic naïve T helper cells were activated by TCR activation in the anti-CD3e/CD28 coated plates under Th2 differentiation condition for 72 hours, rested 42 hours and restimulated by TCR activation using anti-CD3e/CD28 coated plates. 15uM 4m8c was added to the culture media RNA was purified using the Qiagen RNEasy Plus Mini Kit automated using a QIAcube Samples were processed using the TruSeq RNA Sample Prep v2 kit (Illumina) according to the manufacturers instructions with an alteration to use KAPA Hifi polymerase for PCR amplification instead of the one supplied with the kit.
Experimental Factor: cell type
naive T cell
Experimental Factor: compound
none
Experimental Factor: time
0