Back to results
US 2021/0262019 A1Published application
Methods of making gene expression libraries
Assignee10x Genomics, Inc.
InventorsLuigi Jhon Alvarado Martinez, Eswar Prasad Ramachandran Iyer
Life of this patent
Estimating the term…
Abstract
Provided herein are methods of determining a location of a target nucleic acid in a biological sample.
Claims20
A method of identifying a location of a target nucleic acid in a permeabilized biological sample, 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 biological sample;
- (b) ligating a second adaptor sequence to a 3′ end of the cDNA molecule, wherein the step of ligating is performed within the biological sample;
- (c) releasing the cDNA molecule from the target nucleic acid, such that the cDNA 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 specifically to the second adaptor sequence ligated to the cDNA;
- (d) extending a 3′ end of the capture probe using the cDNA 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) all or a part of 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 biological sample.
4 more elements
A method of identifying a location of a target nucleic acid in a permeabilized biological sample, 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 biological sample;
- (b) extending a 3′ end of the cDNA molecule to include a second adaptor sequence, wherein the step of extending is performed within the biological sample;
- (c) releasing the cDNA molecule from the target nucleic acid, such that the cDNA 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 specifically to the second adaptor sequence;
- (d) extending a 3′ end of the capture probe using the cDNA 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 biological sample.
4 more elements
A nucleic acid comprising, in the 5′ to 3′ direction:
- a spatial barcode;
- a sequence complementary to a second adaptor sequence;
- a sequence present in a target nucleic acid;
- and
- a sequence complementary to a first adaptor sequence.
2 more elements
Dependencies are inferred from the claim text.