BOLT-seq – cost and time-efficient construction of a 3′-end mRNA library from unpurified bulk RNA in a single tube

Unraveling the transcriptome—the complete set of RNA molecules in a cell—is pivotal for understanding gene expression and regulatory mechanisms. RNA sequencing (RNA-seq) has emerged as a powerful tool for transcriptome profiling, offering unparalleled accuracy. However, traditional RNA-seq methods come with significant drawbacks, including high costs and limited scalability. In an exciting development, researchers at Yonsei University have introduced a groundbreaking approach called BOLT-seq, offering a highly scalable and cost-effective solution for transcriptomics profiling.

BOLT-seq represents a paradigm shift in transcriptome profiling, bypassing the need for RNA purification and complex library construction procedures. Instead, this innovative method leverages unpurified bulk 3′-end mRNA directly from crude cell lysates. During BOLT-seq, RNA/DNA hybrids undergo tagmentation—a process where DNA fragments are tagged with adapters—without the intermediate steps of second-strand cDNA synthesis and RNA purification. Remarkably, libraries can be constructed in just 2 hours of hands-on time, making BOLT-seq a swift and efficient technique for transcriptome analysis.

Overall BOLT-seq scheme

Fig. 1

Bulk transcriptOme profiling of cell Lysate in a single poT—BOLT-seq. The steps of BOLT-seq are shown schematically. Sequential reactions were carried out in a single well of a 96-well microtiter plate with no intermediate purification steps. After the final library amplification step, products are purified from each well and pooled as desired.

The versatility of BOLT-seq extends beyond its streamlined protocol—it holds immense promise for a wide range of applications in molecular research. One notable application is in drug discovery, where BOLT-seq has been successfully employed to cluster small molecule drugs based on their mechanisms of action and intended targets. By providing rapid and cost-effective transcriptome profiling, BOLT-seq empowers researchers to explore gene expression dynamics in response to various experimental conditions and drug treatments with unprecedented efficiency.

The introduction of BOLT-seq marks a transformative milestone in transcriptomics research, offering a cost-effective and scalable solution for transcriptome profiling. By streamlining library construction and bypassing the need for RNA purification, BOLT-seq empowers researchers to delve deeper into the complexities of gene expression and regulatory networks. As scientists continue to harness the capabilities of BOLT-seq across diverse biological contexts, we can anticipate groundbreaking discoveries that will advance our understanding of cellular processes and pave the way for innovative therapeutic interventions.

Choi J, Hyun J, Hyun J et al. (2024) Cost and time-efficient construction of a 3′-end mRNA library from unpurified bulk RNA in a single tube. Exp Mol Med [Epub ahead of print]. [article]

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