![]() S1-S2, Additional files 2 and 3) and single-cell inputs (Additional file 1: Fig. We altered various experimental parameters of the original SHERRY protocol for both bulk (Additional file 1: Fig. Spiking template-switching oligonucleotides also provides more uniform coverage, but this strategy has limited detection sensitivity and specificity. Although adding random RT primers facilitates the coverage of long transcripts, it requires the removal of ribosomal RNA, which is incompatible with scRNA-seq. SHERRY2 provides high sensitivity and even coverage across gene bodies for scRNA-seqįor scRNA-seq, RNA degradation and incompleteness of reverse transcription (RT) are two major factors that reduce gene detection sensitivity and coverage evenness. In comparison with prevalent RNA-seq methods, SHERRY2 showed higher sensitivity, better concordance with reference data, greater reproducibility between replicates, and superior scalability, allowing the method to be used to process a few thousand single cells per batch and thus reducing the time required to conduct experiments. In this work, we present an optimized method, SHERRY2, which addresses the limitations of SHERRY and is fully compatible with single cells and single nuclei with low RNA content. Īlthough SHERRY was applied to process single cells and achieved less biased quantification of gene expression in comparison with other scRNA-seq methods, the results still exhibited clear coverage bias toward the 3′-ends of transcripts, relatively low sensitivity, and low tolerance to endogenous DNA. With slight modifications, SHERRY could also be applied to various clinical metatranscriptome applications, such as the identification of SARS-CoV-2 and other pathogens. The development of SHERRY was based on the recent discovery that Tn5 transposase can bind and cut RNA/DNA hetero-duplexes directly. We have reported a highly reproducible and rapid library preparation method for RNA-seq, SHERRY, which can be applied to a minute amount of RNA samples. Besides, their complex experimental methods are generally unsuitable for large-scale studies. Single tube-based scRNA-seq approaches can typically produce higher coverage for low-abundance genes, but they still suffer from quantification bias due to insufficient reverse transcription and GC imbalance during amplification. However, large-scale scRNA-seq techniques, commonly operated in micro-droplets or wells, have relatively low sensitivity. Prevalent scRNA-seq methods mainly rely on template switching and pre-amplification of complementary DNA (cDNA). High-quality single-cell RNA-seq (scRNA-seq) data can be used to reveal the kinetic details of gene expression and transitions between cell states or types. Many experimental methods for transcriptome profiling by next-generation sequencing (RNA-seq) have been developed to cover various scales of input samples, ranging from bulk samples to single cells or even subcellular components. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries. SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. Resultsīased on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA.
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