GSM1854547: MB7B7; Mus musculus; RNA-Seq
40 week old Apoe-/- spleens (4-5 spleens/C1 IFC preparation, six independent IFC preparations) were collected, and CD4+ T cells were isolated by negative selection (Stem cell Technologies, Murine CD4+ EasySep kits). After extracellular staining with antibodies, CD4+CD73+PD1+CD25+CCR5+ T cells were isolated by cell sorting, as this population contains a mixture of Th1, Treg, and Th1/Treg cells. Isolated CD4+CD73+PD1+CD25+CCR5+ T cells were loaded onto 5-10um Fluidigm C1 IFCs in C1 suspension reagent to capture single cells, following the manufacturer's protocol. cDNA was prepared and amplified using the SMARTer Ultra Low RNA Kit and Advantage 2 PCR kits (Clontech), following the Fluidigm C1 Auto-prep mRNA seq: RT & Amp script (1772x/1773x) and C1 protocol. The single T cell cDNA yeilds were determined using the Quant-iT PicoGreen dsDNA assay (Life Technologies). 0.1-0.3 ng of cDNA was used to prepare the Nexterra cDNA libraries for illumina sequencing. Nextera XT cDNA libraries were tagmented and indexed using the Nextera XT  DNA Sample Preparation kit, Nextera XT indexing primers (Illumina), and AMPure XP beads (Agencourt), following the Fluidigm C1 protocol and the manufacturer's instructions. 48 indexed libraries were pooled/lane and sequenced with an Illumina HiSeq 2000 (50bp, single ended reads, six lanes total). Base calling and demultiplexing was conducted using CASAVA to generate Fastq files. The Illumina indexing primer sequences are included in the Fastq file name.
GEO Accession
GSM1854547