Abstract
Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.
Highlights
Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity
not-so-random primers (NSRs) are designed to avoid synthesizing Complementary DNA (cDNA) from the ribosomal RNAs (rRNAs) by removing 6-mers that exactly match the rRNA sequences from N6 random primers
RamDA-seq revealed many known and unannotated non-poly(A) transcripts that were dynamically regulated as differentiation progressed, including Neat[1] (Fig. 3)
Summary
Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. SUPeR-seq shows relatively low sensitivity with non-poly(A) RNAs (20–30%)[20], which leaves room for developing scRNA-seq methods with higher sensitivity to non-poly(A) RNAs. In addition, how to reduce the sequence derived from ribosomal RNAs (rRNAs) that accounts for most of the total RNA is a major task for establishing single-cell total RNA-seq. A single-cell total RNA-seq method with both full-length transcript coverage and high sensitivity for non-poly(A) RNAs remains to be developed. It is necessary to simplify an experimental procedure, to use RT with high capture efficiency and to amplify the cDNA as early step as possible To satisfy these conditions, we decided to use a novel RT technology: RT with random displacement amplification (RT-RamDA)
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