Expression profiling by high throughput sequencing
Summary
Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed “TRACE-seq”) are comparable to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis; at the meantime, TRACE-seq enables a one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.
Overall design
mRNA profiles of HEK293T cells were generated by TRACE-seq with different RT primers (random primer, in duplicates; oligodT primer, in duplicates) and by NEBNext Ultra II RNA kit in duplicates with 10ng mRNA as input. In addition, mRNA profiles of HEK293T cells were generated by TRACE-seq with different amounts of total RNA as input (200ng total RNA, in triplicates; 20ng total RNA, in duplicates; 2ng total RNA, in duplicates) and by Smart-seq2 with 20ng and 2ng total RNA as input in duplicates respectively. What's more, differential gene expression analysis between differential and undifferential mESC cells was performed by TRACE-seq and NEBNext Ultra II RNA kit in triplicates respectively.