![]() ![]() S2a), consistent with a previous report that used a 14-nt single-stranded template. When an 18-bp dsDNA linker was included between the upstream T7 promoter and the downstream ssRNA template, we found that T7 IVT was also efficient (Additional file 1: Fig. Here, we tested the efficiency of T7 IVT with a 20-nt ssRNA template as compared to a dsDNA template of the same sequence. T7 in vitro transcription (IVT) of double-stranded DNA (dsDNA) templates to generate antisense RNA (aRNA) has been widely used to achieve linear amplification in single-cell assays. Using LAST-seq, we characterized gene expression noise and transcriptional bursting kinetics, and investigated the regulation of transcriptional activities by topologically associating domains (TADs) in human cells.Įfficient T7 in vitro transcription of single-stranded RNA templates Rather than relying on the inefficient RT/SSS prior to RNA amplification, LAST-seq directly amplifies the original ssRNA molecules in single cells in a linear fashion, achieving a high single-molecule capture efficiency and a low level of technical noise compared to existing scRNA-seq methods. To address this limitation, we developed a new scRNA-seq assay called LAST-seq. S1b), hindering accurate characterization of cell-to-cell variation and gene expression noise. Low RNA capture efficiency also adds a high level of technical noise in the single-cell transcriptome data (Additional file 1: Fig. S1a), undermining the ability to distinguish cell types with a subtle difference in the gene expression level. Low RNA capture efficiency can lead to measurement inaccuracies (Additional file 1: Fig. Regardless of the specific protocol, the inevitable RT/SSS with a limited efficiency in existing scRNA-seq methods ultimately compromises the single-molecule capture efficiency of the original RNA molecules. While RT remains reliant on reverse transcriptase, current scRNA-seq assays employ various SSS strategies with a limited efficiency, including the use of terminal transferase or template switching to create cDNA priming sites for PCR, employing RNase H and DNA Pol to convert the RNA/cDNA hybrid to double-stranded DNA for in vitro transcription, random annealing to the single-stranded cDNA for extension, or Tn5 tagmentation of the RNA/cDNA hybrid. The underlying chemistry, however, remains unchanged and depends on the same fundamental step of RT/SSS prior to the amplification of single-stranded RNA (ssRNA) molecules. Recent technical advances have focused on improving the performance of digital counting by unique molecular identifiers (UMIs), enhancing the cellular throughput while lowering the cost, optimizing individual steps in the protocol, and miniaturization. Single-cell transcriptome analyses have been driven by the invention of scRNA-seq methods. ![]()
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