title: Illumina HiSeq 2000 sequencing; An mRNA-sequencing atlas of mouse CD4+ T cell transcriptomes
design: An mRNA-sequencing atlas of mouse CD4+ T cell transcriptomes
library_construction_protocol: CD4+, CD62L+ cells were purified from DEREG mice with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec). Cells were seeded into anti-CD3 (1 ug/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 ug/ml, clone 37.51, eBioscience) coated 96-well plates, at a density of 250,000-500,000 cells/ml and cultured in a total volume of 200 ul in the presence of recombinant human TGF-beta1 (20ng/ml, Sigma). The cells were gently removed from the activation plate on day 4. The iTreg culture was subjected to FACS sorting for GFP-positive, propidium iodide-negative cells. Poly-(A)+ RNA was purified from ~500,000 cells using the Oligotex kit (Qiagen). The manufacturers protocol was modified slightly to include additional final elution steps resulting in a larger volume. RNA was purified, reverse transcribed and prepared for sequencing described in http://msb.embopress.org/content/7/1/497