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Scheme of spatial-CITE-seq. A cocktail of ADTs is applied to a PFA-fixed tissue section to label a panel of ~200–300 protein markers in situ. Next, a set of DNA barcodes A1–A50 is flowed over the tissue surface in a spatially defined manner via parallel microchannels, and reverse transcription is carried out inside each channel for in-tissue synthesis of cDNAs complementary to endogenous mRNAs and introduced ADTs. Then, a set of DNA barcodes B1–B50 is introduced using another microfluidic device with microchannels perpendicular to the first flow direction and subsequently ligated to barcodes A1–A50, creating a 2D grid of tissue pixels, each of which has a unique spatial address code AB. Finally, barcoded cDNA is collected, purified, amplified and prepared for paired-end NGS sequencing.

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