Methods for spatial analysis using proximity ligation

A method of determining a location of an interaction between a protein and a nucleic acid in a tissue sample, the method comprising:

• (a) contacting the tissue sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;
• (b) attaching a first analyte-binding moiety to the protein, wherein the first analyte-binding moiety is bound to a first oligonucleotide comprising a first barcode that is unique to the interaction between the protein and the first analyte-binding moiety;
• (c) hybridizing a second oligonucleotide to the nucleic acid, wherein the second oligonucleotide comprises a capture probe capture domain sequence;
• (d) hybridizing a third oligonucleotide to the first oligonucleotide and to the second oligonucleotide;
• (e) washing the tissue sample to remove any third oligonucleotides that are unbound to the first oligonucleotide and/or the second oligonucleotide;
• (f) ligating the first oligonucleotide and the second oligonucleotide, thereby generating a ligation product comprising the first barcode and the capture probe capture domain sequence;
• (g) dissociating the third oligonucleotide from the first oligonucleotide and the second oligonucleotide;
• (h) while the capture domain of the capture probe is affixed to the substrate, hybridizing the capture probe capture domain sequence of the ligation product to the capture domain;
• and
• (i) determining (i) the sequence of the spatial barcode or a complement thereof;
• (ii) the sequence of the first barcode or a complement thereof, and (iii) a sequence of the second oligonucleotide that is complementary to the nucleic acid or a complement thereof;
• and using the determined sequences of (i), (ii), and (iii) to identify the location of the interaction between the protein and the nucleic acid in the tissue sample.