Principle behind the double-barcoded spatial epigenomic technology. (A) DNA arrays are printed by depositing first probes in rows, followed by deposition of a second type of probes in columns. The first type of probe is composed of a T7 promoter (T7p) sequence, a unique molecular barcode associated to the row (BCr1, …BCri), and bridge sequence called “Gibson.” The second type of probe presents a complementary Gibson sequence, a unique molecular barcode associated to the column (BCc1, …BCcj), and a complementary mosaic sequence (MOS′). (B) Because both probes were combined during printing, after UV irradiation for immobilization, hybridized probes are elongated with the T4 DNA polymerase, leading to a double-strand molecule presenting the mosaic sequence (MOS) at the free end of the probe. (C) After depositing a tissue section on top of the manufactured DNA array, a first antibody against the protein of interest is incubated, followed by a second antibody against the first antibody, and is finally incubated with the recombinant Protein A–transposase PA-Tn5. (D) Scheme illustrating the loading of the Tn5 into the mosaic sequence retrieved on the printed probes on the DNA array, followed by the chromatin cleavage induced by magnesium chloride, leading to the formation of hairpin-like DNA structures. (E) Scheme illustrating the molecular biology steps required for generating a copy of the genomic DNA captured by the DNA probes, followed by their amplification for Illumina massive parallel DNA sequencing.
