title: Th17 rep2
organism: Mus musculus
description: Protocols: 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, RD 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
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-04-05T17:04:12Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T17:48:51Z
attributes.tag: External Id
attributes.value: SAMEA2536557
attributes.tag: INSDC center name
attributes.value: EBI
attributes.tag: INSDC first public
attributes.value: 2015-04-05T17:04:12Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T17:48:51Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2582:Th17 rep2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: T-helper 17 cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: sample name
attributes.value: E-MTAB-2582:Th17 rep2
attributes.tag: scientific_name
attributes.value: Mus musculus