Single-cell transcriptome analysis gives insight into the heterogeneity of neighboring cells in tissues or cell cultures. It has been shown that the variability of cells is important for biological function. However, cell variability can be an indication of disease (e.g., cancer). In order to elucidate cell variability in health and disease, single-cell transcriptomes can be analyzed by new next-generation sequencing (NGS) platforms. NGS analysis currently requires a few hundred nanograms of RNA. Consequently, accurate whole-transcriptome amplification is required to analyze the transcriptome of a single cell by NGS.
This protocol describes the entire workflow from cells to NGS, including cell quality testing, cell lysis, gDNA removal, whole-transcriptome amplification, and NGS library preparation. Recommendations for WTA quality control are given and optional protocols for using purified RNA for WTA and WTA purification are described. (read more...)