Extended Data Fig. 1: Chemistry workflow of spatial-ATAC-seq.

A tissue section on a standard aminated glass slide was lightly fixed with formaldehyde. Then, Tn5 transposition was performed at 37 °C, and the adapters containing ligation linker 1 were inserted to the cleaved genomic DNA at transposase accessible sites. Afterwards, a set of DNA barcode A solutions were introduced by microchannel-guided flow delivery to perform in situ ligation reaction for appending a distinct spatial barcode Ai (i = 1–50) and ligation linker 2. Then, a second set of barcodes Bj (j = 1-50) were introduced using another set of microfluidic channels perpendicularly to those in the first flow barcoding step, which were subsequently ligated at the intersections, resulting in a mosaic of tissue pixels, each containing a distinct combination of barcodes Ai and Bj (i = 1–50, j = 1–50). After DNA fragments were collected by reversing cross-linking, the library construction was completed during PCR amplification.