US 12,071,655 B2
Methods, compositions, kits, and systems for enhancing analyte capture for spatial analysis
David Sukovich, Oakland, CA (US); Hanyoup Kim, Foster City, CA (US); Jerald Sapida, San Leandro, CA (US); Spontaneous Rose McKnight Russell, San Ramon, CA (US); Layla Katiraee, Castro Valley, CA (US); and Augusto Manuel Tentori, Dublin, CA (US)
Assigned to 10x Genomics, Inc., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on May 3, 2023, as Appl. No. 18/311,605.
Application 18/311,605 is a continuation of application No. PCT/US2022/031923, filed on Jun. 2, 2022.
Claims priority of provisional application 63/196,435, filed on Jun. 3, 2021.
Prior Publication US 2023/0295699 A1, Sep. 21, 2023
Int. Cl. C12Q 1/6841 (2018.01); C12N 15/10 (2006.01)
CPC C12Q 1/6841 (2013.01) [C12N 15/1065 (2013.01)] 26 Claims
OG exemplary drawing
 
1. A method for determining a location of multiple analytes in a biological sample, the method comprising:
(a) providing a first substrate and the biological sample mounted thereon, wherein the first substrate comprises a plurality of first capture probes, wherein a first capture probe of the plurality of first capture probes comprises (i) a first spatial barcode and (ii) a first capture domain;
(b) aligning a second substrate on the opposite side of the first substrate relative to the biological sample, thereby sandwiching the biological sample between the first and the second substrate, wherein the second substrate comprises a plurality of second capture probes, wherein a second capture probe of the plurality of second capture probes comprises (i) a second spatial barcode and (ii) a second capture domain;
(c) adding a reagent medium comprising a permeabilization buffer to the biological sample prior to aligning, thereby promoting migration of a first intermediate agent and a second analyte or a second intermediate agent from the biological sample to the first substrate and/or the second substrate, wherein the first intermediate agent is generated by hybridizing a first probe and a second probe to a first analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to adjacent sequences of the first analyte, and wherein the second probe comprises a first capture probe binding domain; and coupling the first probe and the second probe, thereby generating a first connected probe;
(d) hybridizing the first intermediate agent to the first capture domain, and hybridizing the second analyte or the second intermediate agent to the second capture domain; and
(e)
(A) determining (i) the sequence of the first spatial barcode, or a complement thereof, and (ii) all or a portion of the sequence of the first intermediate agent, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the first analyte in the biological sample, and
(B) determining (iii) the sequence of the second spatial barcode, or a complement thereof, and (iv) all or a portion of the sequence of the second analyte or the second intermediate agent, or a complement thereof, and using the sequences of (iii) and (iv) to determine the location of the second analyte in the biological sample.