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bioRxiv · 10.1101/2025.08.20.671269

Improving RNA-seq protocols

Pfoertner, F.·Briem, E.·Enard, W.·Richter, D.
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Abstract

Bulk and single-cell RNA-seq are powerful tools for transcriptomic analysis, providing insights into many aspects of molecular and cellular phenotypes. Costs constrain the amount of biological insight obtainable within a given budget, and as sequencing prices decline, efficient library protocols have become a decisive factor. In this study, we introduce an approach to systematically optimize the number of usable reads that RNA-seq protocols generate. We applied this "funnel strategy" to prime-seq, an early-barcoding bulk RNA-seq protocol, by systematically testing critical protocol steps totaling 1080 samples in 49 libraries. This resulted in the optimized prime-seq2 protocol that increases the number of usable reads by 60 % and improves one of the most cost-efficient bulk RNA-seq protocols available. Our study also suggests that monitoring the filtering of usable reads can serve as a valuable quality control for many RNA-seq protocols and sheds light on the complexity of the conditions and interactions that shape RNA-seq library composition and their interpretation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/671269v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1bf102dorg.highwire.dtl.DTLVardef@bf36bforg.highwire.dtl.DTLVardef@1a3600aorg.highwire.dtl.DTLVardef@f63470_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Pfoertner, F., Briem, E., Enard, W., Richter, D.. 2025-08-24. Improving RNA-seq protocols. https://doi.org/10.1101/2025.08.20.671269

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