An improved method for the highly specific detection of transcription start sites

Precise detection of the transcriptional start site (TSS) is a key for characterizing transcriptional regulation of genes and for annotation of newly sequenced genomes. Here, we describe the development of an improved method, designated 'TSS-seq2.' This method is an iterative improvement of TSS-seq, a previously published enzymatic cap-structure conversion method to detect TSSs in base sequences. By modifying the original procedure, including by introducing split ligation at the key cap-selection step, the yield and the accuracy of the reaction has been substantially improved. For example, TSS-seq2 can be conducted using as little as 5 ng of total RNA with an overall accuracy of 96%; this yield a less-biased and more precise detection of TSS. We then applied TSS-seq2 for TSS analysis of four plant species that had not yet been analyzed by any previous TSS method. Graphical Abstract

Automated summary

TSS-seq2 is an improved method for detecting TSS, with higher accuracy and less bias compared to previous methods, achieved by introducing split ligation and other modifications. It can be conducted with as little as 5 ng of RNA and has been successfully applied to TSS analysis of four plant species.