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Barcoded AOCs are bound to their respective target proteins on cells (1). DNApixel set A then hybridizes to the universal BS1 sequence of several proximal AOCs, followed by gap-fill ligation to incorporate the UPI sequence of each DNApixel and a common pixel BS2, generating UPI-A-based spatial zones (2). Similarly, DNApixel set B hybridizes to the extended AOCs, adding UPI-B (3). The final product is amplified by PCR using REV and FWD primer sites and sequenced.

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