Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low -to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of -1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzy-matic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was imple-mented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell -cell communication after nerve ligation.

Automated summary

This study introduces a stamping method for fabricating polony gels, reducing the cost and time of barcode sequencing, and developing a single-cell spatial transcriptomic assay. This method offers high resolution and RNA capture efficiency for mapping and analyzing cell-cell communication in tissues.