Native elongating transcript sequencing (NET-seq).

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Advancements in sequencing technologies enable high-resolution observation of genomic activity and gene expression.
• Understanding gene expression regulation requires precise measurement of RNA polymerase activity.
• Existing methods may lack the resolution or directness to capture nascent transcripts effectively.

Purpose of the Study:

• To describe Native Elongating Transcript Sequencing (NET-seq), a novel high-resolution methodology.
• To detail a protocol for capturing nascent RNA transcripts and associated RNA polymerase.
• To enable quantitative measurement of RNA polymerase density across the genome with single-nucleotide precision.

Main Methods:

• NET-seq captures nascent RNA transcripts directly from live Saccharomyces cerevisiae cells within the DNA-RNA-RNAP ternary complex.
• A protocol is provided for creating DNA libraries from these nascent RNA transcripts.
• Next-generation sequencing of the DNA library reveals the identity and abundance of transcript 3' ends.

Main Results:

• Deep sequencing of the DNA library provides a quantitative measure of RNA polymerase (RNAP) density.
• The method achieves single-nucleotide precision in quantifying RNAP distribution.
• The protocol's high efficiency is critical for the quantitative nature of the NET-seq dataset.

Conclusions:

• NET-seq offers a powerful approach to map RNAP density genome-wide at unprecedented resolution.
• The described protocol allows for direct observation of transcriptional activity in live cells.
• Successful implementation relies on careful monitoring of critical protocol steps for high efficiency.