STOMP-seq: early multiplexing for high-throughput SMART RNA-sequencing

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.