title: GSM1942836: EAE-IL17A-CNS_130124_CNS_IL17a_SC65_58; Mus musculus; RNA-Seq
library_construction_protocol: At the peak of disease, 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 mm), 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+. Cell lysis and SMART-Seq (Ramskold et al., 2012) whole transcriptome amplification (WTA) was performed on the C1 chip using the Fluidigm C1 Single-Cell Auto Prep System (C1 System) with the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech). WTA products were harvested from the C1 chip, and cDNA libraries were prepared using Nextera XT DNA Sample preparation reagents (Illumina) as per manufacturer’s recommendations, with minor modifications. Specifically, reactions were run at ¼ the recommended volume, the tagmentation step was extended to 10 minutes, and the extension time during the PCR step was increased from 30s to 60s. After the PCR step, all 96 samples were pooled without library normalization, cleaned twice with 0.9x AMPure XP SPRI beads (Beckman Coulter), and eluted in buffer TE. The pooled libraries were quantified using Quant-IT DNA High-Sensitivity Assay Kit (Invitrogen) and examined using a high sensitivity DNA chip (Agilent). Finally, samples were sequenced deeply using either a HiSeq 2000 or a HiSeq 2500 sequencer.
identifiers.namespace: GEO
identifiers.id: GSM1942836