Method for generating a three-dimensional nucleic acid containing matrix

A method for analysis, comprising:

• (a) providing a biological sample comprising a plurality of cells, wherein said plurality of cells comprises a plurality of nucleic acids, wherein each of said plurality of nucleic acids has a three dimensional (3D) spatial location within said biological sample;
• (b) contacting said biological sample with a plurality of padlock probes to bind at least some padlock probes comprising a plurality of target-specific barcode sequences to at least some of said plurality of nucleic acids in said biological sample, wherein each padlock probe of said at least some padlock probes comprises a target-specific barcode sequence of said target-specific barcode sequences;
• (c) ligating at least some of said padlock probes bound to said nucleic acids to generate circularized padlock probes;
• (d) using said circularized padlock probes to generate rolling circle amplicons in said biological sample;
• (e) generating a three-dimensional (3D) matrix comprising amplicons of said rolling circle amplicons, wherein said amplicons are covalently attached to said 3D matrix, and wherein said 3D matrix maintains a relative 3D spatial location of said amplicons;
• and
• (f) sequencing said plurality of target-specific barcode sequence in said biological sample.