Compositions and methods of making gene expression libraries

A method of determining a location of a target nucleic acid in a biological sample, the method comprising:

• (a) contacting the biological sample with an array comprising a feature, wherein the feature comprises an attached first and second probe, wherein:
• a 5′ end of the first probe is attached to the feature;
• the first probe comprises in a 5′ to a 3′ direction:
• a spatial barcode and a capture domain which binds to the target nucleic acid;
• a 5′ end of the second probe is attached to the feature;
• a 3′ end of the second probe is reversibly blocked;
• and
• the second probe comprises a poly(GI) capture domain;
• (b) binding the first probe to the target nucleic acid and extending the first probe to add a sequence that is complementary to a portion of the target nucleic acid to generate a first extended probe;
• (c) ligating an adapter to the 5′ end of the target nucleic acid bound to the first extended probe;
• (d) adding a sequence complementary to the adapter to the 3′ end of the first extended probe;
• (e) after (d), adding non-templated cytosines to the 3′ end of the first extended probe to generate a poly(C) sequence, wherein the poly(C) sequence binds to the poly(GI) capture domain of the second probe;
• (f) unblocking the 3′ end of the second probe and extending the 3′ end of the second probe to add a sequence comprising a sequence in the target nucleic acid and a sequence that is complementary to the spatial barcode;
• (g) cleaving a region of the second probe at a cleavage site that is 5′ to the poly(GI) capture domain, thereby releasing the second probe from the feature;
• and
• (h) determining (i) the sequence of the spatial barcode or a complement thereof, and (ii) all or a part of the sequence of the target nucleic acid, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the target nucleic acid in the biological sample.