title: GSM1943405: Tgfb1_IL6-IL17A_POS_48hr_130828_48h_t16_il17pos_SC28_185; Mus musculus; RNA-Seq
library_construction_protocol: Cells 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: GSM1943405