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

RNA-sequencing provides high-dimensional, quantitative descriptions of the physiologic state of cells, tissues, organs, and whole organisms. RNA-Seq protocols based on 96 and 364-well plates allow for a wider range of experimental designs compared to droplet sequencing methods, but are less scalable due to the practical challenges of plate-based liquid handling. Here, we present STOMP-seq, an RNA-Seq method that extends Smart-seq2 to include sample barcodes on the 5' end of amplified transcript. The additional barcodes allow samples to be pooled immediately after reverse transcription, enabling a 12-fold multiplexing strategy. In this way, STOMP-seq can reduce liquid handling complexity and enzyme costs several-fold for both manual and robotic library preparation approaches, while improving library complexity and coverage over Smart-seq2. Together, these advantages combine to support new high-throughput experimental designs, which we demonstrate by performing a population-scale, multi-generational study of gene-expression heritability.