Native Elongating Transcript Sequencing (NET‐seq)

Advances in sequencing technology have led to the development of many high-resolution methodologies that observe genomic activity and gene expression. This unit describes such an approach, native elongating transcript sequencing (NET-seq), which reveals the density of RNA polymerase across the Saccharomyces cerevisiae genome with single-nucleotide resolution. A procedure for capturing nascent RNA transcripts directly from live cells through their association with the DNA-RNA-RNAP ternary complex is described. A protocol to create DNA libraries from the nascent RNA, allowing the identity and abundance of the 3' end of purified transcripts to be revealed by next generation sequencing, is also provided. By deep sequencing this DNA library, a quantitative measure of RNAP density with single-nucleotide precision is obtained. The quantitative nature of the NET-seq dataset relies on the high efficiency of many steps in the protocol. The steps that are most critical are described with suggestions for monitoring their success.