title: GSM1942797: EAE-CNS-IL17A/GFP+ (population, rep3); Mus musculus; RNA-Seq
library_construction_protocol: At the peak of disease (EAE), T cells were collected from the draining LNs and the CNS. For isolation from the CNS, mice were perfused through the left ventricle of the heart with cold PBS. The brain and the spinal cord were flushed out with PBS by hydrostatic pressure. CNS tissue was minced with a sharp razor blade and digested for 20 min at 37°C with collagenase D (2.5 mg/ml; Roche Diagnostics) and DNaseI (1 mg/ml; Sigma). Mononuclear cells were isolated by passage of the tissue through a cell strainer (70 μm), followed by centrifugation through a Percoll gradient (37% and 70%). After removal of mononuclear cells, the lymphocytes were washed, stained and sorted for CD3 (Biolegend), CD4 (Biolegend), 7AAD and IL-17A-GFP+. Population controls were generated by extracting total RNA using a RNeasy plus Micro RNA kit (Qiagen) according to manufacturer’s recommendations. Subsequently, 1 μL of RNA in water was added to 2  μL of lysis reaction mix, thermocycled using cycling conditions I described in Supplementary Experimental Procedures in corresponding publication. Next, 4  μL of the RT Reaction Mix were added, and the mixture was thermocycled using cycling conditions II. Finally, 1  μL of the total RT reaction was added to 9 μL of PCR mix, and that mixture was thermocycled using cycling conditions III. Products were quantified, diluted to 0.125 ng/ μL, and libraries were prepared, cleaned, and tested as described in Supplementary Experimental Procedures in corresponding publication.
identifiers.namespace: GEO
identifiers.id: GSM1942797