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US 11,891,654 B2Granted
Methods of making gene expression libraries
Assignee10x Genomics, Inc.
InventorsLuigi Jhon Alvarado Martinez, Eswar Prasad Ramachandran Iyer
Life of this patent
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Abstract
Provided herein are methods of determining a location of a target nucleic acid in a biological sample.
Claims16
A method of identifying a location of a target nucleic acid in a permeabilized tissue section, the method comprising:
- (a) generating a cDNA molecule comprising a sequence that is substantially complementary to the target nucleic acid using a reverse transcription primer comprising (i) a sequence that is substantially complementary to a portion of the target nucleic acid and (ii) a first adaptor sequence, wherein the step of generating the cDNA molecule occurs within the permeabilized tissue section;
- (b) ligating a second adaptor sequence to a 3′ end of the cDNA molecule, wherein the step of ligating is performed within the tissue section;
- (c) releasing the cDNA molecule ligated to the second adaptor sequence from the target nucleic acid in the tissue section, such that the second adaptor sequence contacts an array, wherein the array comprises an attached capture probe comprising in a 5′ to a 3′ direction:
- (i) a spatial barcode and (ii) a capture domain that hybridizes to the second adaptor sequence ligated to the cDNA molecule;
- (d) extending a 3′ end of the capture probe using the cDNA molecule as a template;
- and
- (e) determining (i) all or a part of the sequence of the target nucleic acid, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target nucleic acid in the permeabilized tissue section.
4 more elements
A method of identifying a location of a target nucleic acid in a permeabilized tissue section, the method comprising:
- (a) generating a cDNA molecule comprising a sequence that is substantially complementary to the target nucleic acid using a reverse transcription primer comprising (i) a sequence that is substantially complementary to a portion of the target nucleic acid and (ii) a first adaptor sequence, wherein the step of generating the cDNA molecule occurs within the permeabilized tissue section;
- (b) extending a 3′ end of the cDNA molecule to include a second adaptor sequence thereby generating an extended cDNA molecule, wherein the step of extending is performed within the tissue section;
- (c) releasing the extended cDNA molecule from the target nucleic acid, such that the extended cDNA molecule contacts an array, wherein the array comprises an attached capture probe comprising in a 5′ to a 3′ direction:
- (i) a spatial barcode and (ii) a capture domain that binds to the second adaptor sequence of the extended cDNA molecule;
- (d) extending a 3′ end of the capture probe using the extended cDNA molecule as a template;
- and
- (e) determining (i) all or a part of the sequence of the target nucleic acid, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target nucleic acid in the permeabilized tissue section.
4 more elements
Dependencies are inferred from the claim text.