Systems and methods for analyzing tissue samples

A method for biological analysis, comprising:

• (a) providing a planar surface, wherein:
• the planar surface comprises glass, plastic, or a combination thereof,
• the planar surface further comprises a plurality of capture sites,
• the plurality of capture sites are arranged in a two-dimensional pattern on the planar surface,
• the plurality of capture sites are disposed within or adjacent to one or more hydrogel structures,
• a capture site of the plurality of capture sites comprises a dimension of about 1 micron to about 20 microns,
• each capture site of the plurality of capture sites comprises a plurality of single-stranded oligonucleotides,
• the plurality of single-stranded oligonucleotides are coupled to the planar surface,
• at least a subset of the plurality of single-stranded oligonucleotides comprise:
• i) a capture sequence comprising a plurality of thymine bases that are configured to bind to poly-A sequences,
• ii) a unique molecular identifier,
• iii) a barcode associated with a location on the planar surface, and
• iv) a sequencing primer,
• each oligonucleotide of the subset of the plurality of single-stranded oligonucleotides comprises at least 50 nucleotides,
• a tissue sample is disposed on or adjacent to the planar surface,
• the tissue sample comprises a plurality of cells, and
• the tissue sample is disposed within or adjacent to a hydrogel structure of the one or more hydrogel structures;
• (b) imaging at least a portion of the tissue sample, wherein the imaging comprises bright-field imaging, phase-contrast imaging, fluorescence imaging, or a combination thereof;
• (c) releasing nucleic acids from the tissue sample;
• (d) capturing at least a subset of the nucleic acids on at least the subset of the single-stranded oligonucleotides, thereby obtaining the captured nucleic acids, wherein the captured nucleic acids are immobilized on the planar surface;
• and
• (e) extending at least a subset of the captured nucleic acids using at least portions of the single-stranded oligonucleotides as templates, thereby obtaining the extended nucleic acids;
• (f) amplifying the extended nucleic acids, thereby generating amplicons, wherein:
• the amplification is performed using primers that comprise sequencing adaptors;
• (g) sequencing at least a portion of an amplicon of the amplicons, a derivative of the amplicon, a complement of the amplicon, or a combination thereof;
• and
• (h) associating a sequence obtained from the sequencing in (g) and imaging data from the imaging in (b) with a cell of the plurality of cells.