Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing

Abstract

Single-cell analysis of gene expression is increasingly important for the analysis of complex tissues, including cancer, developing organs and adult stem cell niches. Here we present a detailed protocol for quantitative gene expression analysis in single cells, by the sequencing of mRNA 5' ends. In all, 96 cells are lysed, and their mRNA is converted to cDNA. By using a template-switching mechanism, a bar code and an upstream primer-binding sequence are introduced simultaneously with reverse transcription. All cDNA is pooled and then prepared for 5' end sequencing, including fragmentation, adapter ligation and PCR amplification. The chief advantage of this approach is the great reduction in cost and time, afforded by the early bar-coding strategy. Compared with previous methods, it is more suitable for large-scale quantitative analysis, as well as for the characterization of transcription start sites, but it is unsuitable for the detection of alternatively spliced transcripts. Sample preparation takes 3 d, and two sets of 96 cells can be prepared in parallel. Finally, the sequencing and data analysis can take an additional 4 d altogether.

Rewired Metabolism in Drug-resistant Leukemia Cells

“…Samples of each line were prepared in triplicate for expression analysis by RNAseq. A detailed protocol for single cell RNAseq analysis has previously been reported (18,19) and a very similar method was adopted in this work. Briefly, samples were prepared by adding 10 ng of RNA into each well of a 96-well PCR plate preloaded with lysis buffer.…”

Topographical transcriptome mapping of the mouse medial ganglionic eminence by spatially-resolved RNA-seq

“…We have previously developed a method, termed single-cell tagged reverse transcription (STRT), that enables the characterization of single-cell transcriptional landscapes by highly multiplexed RNA-sequencing (RNA-seq) [10,11]. As an initial step towards genomewide transcriptome imaging of tissue sections, we have adapted the STRT method to laser microdissected tissue samples.…”

Switching on sex: transcriptional regulation of the testis-determining geneSry

“…Finally, promoterenhancer interactions can be captured in situ through the use of chromosome conformation capture methods such as Hi-C (Lieberman-Aiden et al, 2009). Whereas ChIP-seq experiments continue to require in the order of 10 4 cells (Adli and Bernstein, 2011), presenting a formidable target when working with embryonic gonads, protocols exist to perform CAGE (Islam et al, 2012) and Hi-C (Nagano et al, 2013) on single cells. Not only does this scale make these strategies more amenable to the small samples • available from nascent organs, but, if applied to groups of cells from different parts of the developing gonad, it also provides the potential for understanding intra-tissue transcriptional dynamics, such as the wave pattern of Sry expression.…”