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bioRxiv · 10.1101/2025.03.28.645277

STOMP-seq: early multiplexing for high-throughput Smart-seq2 RNA-sequencing.

Eder, M.·Stroustrup, N.
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Abstract

RNA-sequencing provides high-dimensional, quantitative measurements of the states of cells, tissues, organs, and whole organisms. Plate-based RNA-seq protocols allow for a wider range of experimental designs than droplet sequencing methods, but are less scalable due to the practical challenges of plate-based liquid handling. Here, we present STOMP-seq, a method that extends SMART RNA-seq protocols, like Smart-seq2 and Smart-seq3, to include sample-identifying barcodes on the 5 end of each amplified transcript. These barcodes allow samples to be pooled immediately after reverse transcription, enabling a 12-fold multiplexing strategy that reduces liquid handling complexity and enzyme costs several-fold. Suitable for both manual and robotic library preparation approaches, STOMP-seq reduces protocol execution times four-fold while improving library complexity and coverage. Together, these advantages combine to make possible new large-scale experimental designs, in particular population-scale sequencing projects like the multi-generational study of gene-expression heritability presented here. STOMP-seq offers a "drop-in" replacement for Smart-seq2 and Smart-seq3, removing practical barriers that currently limit the quality and scope of plate-based transcriptomic data. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/645277v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@83f1b4org.highwire.dtl.DTLVardef@717012org.highwire.dtl.DTLVardef@174e34forg.highwire.dtl.DTLVardef@f790b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Eder, M., Stroustrup, N.. 2025-03-28. STOMP-seq: early multiplexing for high-throughput Smart-seq2 RNA-sequencing.. https://doi.org/10.1101/2025.03.28.645277

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