Seq-Well is a high-throughput, picowell-based single-cell RNA-seq technology that can be used to simultaneously profile the transcriptomes of thousands of cells (Gierahn et al. Nat Methods 14(4):395-398, 2017). Relative to its reverse-emulsion-droplet-based counterparts, Seq-Well addresses key cost, portability, and scalability limitations. Recently, we introduced an improved molecular biology for Seq-Well to enhance the information content that can be captured from individual cells using the platform. This update, which we call Seq-Well S3 (S3: Second-Strand Synthesis), incorporates a second-strand-synthesis step after reverse transcription to boost the detection of cellular transcripts normally missed when running the original Seq-Well protocol (Hughes et al. Immunity 53(4):878-894.e7, 2020). This chapter provides details and tips on how to perform Seq-Well S3, along with general pointers on how to subsequently analyze the resultant single-cell RNA-seq data.
Keywords: Picowells; RNA-Seq; Seq-Well; Single-cell RNA sequencing; Single-cell genomics; Systems biology; Transcriptomics; scRNA-seq.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.