Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation.
Here, an international team of researchers from China and the United States, use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. They show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.
Tn5 tagmentation activity on double-stranded hybrids and the experimental process of SHERRY.
(A) RNase H-like (RNHL) domain alignment of Tn5 (TN5P_ECOLX), RNase H (RNH_ECOLI), and MMLV reverse transcriptase (POL_MLVMS). Active residues in the RNHL domains are labeled in bright yellow. Orange boxes represent other domains. (B) Superposition of the RNHL active sites in these three enzymes. Protein Data Bank IDs are 1G15 (RNase H), 2HB5 (MMLV), and 1MUS (Tn5). (C) Putative mechanism of Tn5 tagmentation of a RNA/DNA hybrid. Crooked arrows represent nucleophilic attacks. (D) Size distribution of mRNA/DNA hybrids with and without Tn5 tagmentation and after amplification with index primers. (E) Workflow of sequencing library preparation by SHERRY. The input can be a lysed single cell or extracted bulk RNA. After reverse transcription with oligo-dT primer, the hybrid is directly tagmented by Tn5, followed by gap-repair and enrichment PCR. Wavy and straight gray lines represent RNA and DNA, respectively. Dotted lines represent the track of extension step.