Methods of detecting analytes
Assignee10x Genomics Sweden AB
InventorsJonas Frisen, Patrik Stahl, Joakim Lundeberg
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Abstract
Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.
Claims47
A method for detecting mRNA from a tissue section, wherein the method comprises:
- (a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized on the feature, wherein the plurality of capture probes comprise the following domains:
- (i) a positional domain comprising a nucleotide sequence unique to the feature;
- and
- (ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
- (b) permeabilizing the tissue section to release mRNA from the tissue section;
- (c) hybridizing released mRNA from the tissue section to the capture domains of the capture probes;
- (d) extending the capture probes using the hybridized mRNA as an extension template to generate cDNA;
- wherein the cDNA is tagged by the positional domain;
- (e) generating second strand cDNA using a template switching primer;
- (f) releasing the second strand cDNA from the features of the array;
- (g) determining the sequence of the second strand cDNA, or a complement thereof, and
- (h) correlating the sequence of the second strand cDNA, or the complement thereof, to a location in the tissue section.
A method for spatially tagging mRNA from a tissue section, wherein the method comprises:
- (a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized at the feature, wherein the plurality of capture probes comprise the following domains:
- (i) a positional domain comprising a nucleotide sequence unique to the feature;
- and
- (ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
- (b) permeabilizing the tissue section to release mRNA from the tissue section;
- (c) hybridizing released mRNA from the tissue section to the capture domains of the plurality of capture probes;
- and
- (d) extending the plurality of capture probes using the hybridized mRNA as an extension template to generate cDNA;
- wherein the cDNA is tagged by the positional domain;
- (e) generating second strand cDNA using a template switching primer;
- and
- (f) releasing the second strand cDNA from the features of the array.
A method for detecting mRNA in a tissue section, wherein the method comprises:
- (a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized on the feature, wherein the plurality of capture probes comprise the following domains:
- (i) a positional domain comprising a nucleotide sequence unique to the feature;
- and
- (ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
- (b) permeabilizing the tissue section to release mRNA from the tissue section;
- (c) hybridizing released mRNA from the tissue section to the capture domains of the plurality of capture probes;
- (d) extending the capture probes using the hybridized mRNA as an extension template to generate cDNA;
- wherein the cDNA is tagged by the positional domain;
- (e) introducing a polynucleotide tail to the cDNA by terminal transferase activity;
- (f) generating a complementary strand of the tagged cDNA;
- (g) releasing at least part of the tagged cDNA and/or the complement thereof, from the features of the array;
- wherein the part includes the positional domain, or a complement thereof;
- (h) determining the sequence of the tagged cDNA, or the complement thereof;
- and
- (i) correlating the sequence of the tagged cDNA, or the complement thereof, to a location in the tissue section.
Dependencies are inferred from the claim text.