title: RNA-seq of mouse TH2 cells
design: Mouse CD4+ T cells were isolated from the spleen and lymph nodes using the EasySep Mouse CD4 Positive Selection Kit II (Stemcell Technologies). Nave (CD25- CD44hi CD62Llo) CD4+ T cells were sorted by flow cytometry from the bead purified CD4+ T cells. The nave CD4+ T cells were resuspended in Clicks medium (Irvine Scientific) at 1 million cells per mL, and then plated on Day 0 in 24 well plates coated with goat-hamster IgG antibody (200ng/ml; MP Biomedicals) with the addition of soluble anti-CD3 (1g/ml; 145-2C11) and anti-CD28 (1g/ml; 37.51) from Bio X Cell. Polarizing conditions for TH2: hIL2 (100U/ml; PeproTech), mIL-4 (20ng/ml; Biolegend), anti-IFN- and anti-IL-12 (5g/ml; Bio X Cell).  Where indicated, Rosi was dissolved in DMSO and added to a final concentration of 10M on Day 0 and Day 3 of 4-5 day cultures. RNA-sequencing libraries were prepared from 100 ng total RNA as per standard Illumina protocols.