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

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.