C1 CAGE and RNA-seq quantification of single-cell gene expression during TGF-β stimulation of A549 cells.

C1 CAGE is a method to quantify single-cell gene expression by transcriptome sequencing on the Illumina platform. This method generates random-primed cDNAs, which allows detection of 5'-end transcripts to map transcription start sites (TSS) at single-nucleotide resolution. This method is performed in the microfluidics chips of the Fluidigm C1 system. The single cell expression data is derived from human A549 adenocarcinomic human alveolar basal epithelial cells stimulated with TGF-β for 0, 6 and 24 h. We used the "Rev A" and "Rev B" version of C1 CAGE, where ERCC spikes are diluted 200 and 20,000 times, respectively, and the standard C1 RNA-seq protocol (PN 100-7168 I1). The stimulations were ran in triplicates, which were multiplexed to control for chip bias using a color-coding strategy: cells stimulated at 0, 6 and 24 hrs were stained with different calcein dyes and pooled prior to loading into the C1 system. Raw images of capture sites were taken using Cellomics ArrayScan VTI High Content Analysis Reader (single images) or IN Cell Analyzer 6000 (z-stack).