Single-Cell RNA-Seq of Mouse Dendritic Cells

Bone marrow derived mouse DCs were prepared as previously described^{18 (link)} and stimulated with pathogenic stimuli for specified time periods. The C₁ Single-Cell Auto Prep System (Fluidigm) was used to perform SMARTer (Clontech) whole transcriptome amplification (WTA)^{15 ,16 (link),19} on up to 96 individual cells. WTA products were then converted to Illumina sequencing libraries using Nextera XT (Illumina)¹⁵ . RNA-Seq libraries were also made from 10,000 cells from each parent population (population control). Each sample was sequenced on an Illumina HiSeq 2000 or 2500, and expression estimates (transcripts per million; TPM) for all UCSC-annotated mouse genes were calculated using RSEM^{36 (link)}. Data was further analyzed as described in the SI. Additional experiments were performed using RNA-FISH (Panomics), “on-chip” isolated stimulation, knockout mice, secretion blockers (GolgiPlug, BD Biosciences), protein synthesis blockers (Cycloheximide, Sigma), and recombinant cytokines. Full Methods and any associated references are provided in SI. Data are deposited in GEO under accession number GSE48968.