title: Illumina HiSeq 2000 paired end 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 with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec) and further purification was performed by FACS using anti-CD62L and anti-CD4 Fab fragments. 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 IL-6 (30ng/ml, Immunotools), recombinant human TGF-beta1 (5ng/ml, Sigma), recombinant murine IL-1beta (10ng/ml, Immunotools), recombinant murine IL-23 (10ng/ ml, R&D systems), neutralizing anti-IL4 (5ug/ml, clone 11B11, eBioscience), neutralizing anti-IFN-gamma (5ug/ml, clone XMG1.2, eBioscience), and neutralizing anti-IL2 (5ug/ml, clone JES6-5H4, eBioscience). Cells were labelled with anti-CCR6-APC (1:200, clone 29-2L17, BioLegend), anti-CD8a-FITC (1:1500, clone 53-6.7, eBioscience), and propidium iodide. FACS sorting was then performed to select FITC-negative, APC-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