US 12,325,877 B2
Compositions and methods for gene expression library analysis
Patrick J. Marks, Paris (FR); and Michael Schnall-Levin, San Francisco, CA (US)
Assigned to 10x Genomics, Inc., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Oct. 24, 2024, as Appl. No. 18/926,208.
Application 18/926,208 is a continuation of application No. 18/589,354, filed on Feb. 27, 2024.
Application 18/589,354 is a continuation of application No. PCT/US2023/021925, filed on May 11, 2023.
Claims priority of provisional application 63/340,730, filed on May 11, 2022.
Prior Publication US 2025/0051842 A1, Feb. 13, 2025
Int. Cl. C12Q 1/6869 (2018.01); C12N 15/10 (2006.01); C12Q 1/48 (2006.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12N 15/1086 (2013.01); C12Q 1/485 (2013.01); C12Q 1/6874 (2013.01)] 20 Claims
 
1. A method of gene expression analysis, comprising:
a) contacting a first RNA analyte and a second RNA analyte in a cell or tissue sample with a first probe and a second probe, respectively, to generate a first amplicon and a second amplicon of a library of amplicons,
wherein the first probe is a first padlock probe comprising a 3′ hybridization region and a 5′ hybridization region complementary to the first RNA analyte in the arms of the first padlock probe and the second probe is a second padlock probe comprising a 3′ hybridization region and a 5′ hybridization region complementary to the second RNA analyte in the arms of the second padlock probe,
wherein the first amplicon comprises i) a first sequencing priming site and ii) a first identifier sequence, and the second amplicon comprising i) a second sequencing priming site different from the first sequencing priming site and ii) a second identifier sequence,
wherein the first identifier sequence is complementary to at least a portion of the 5′ hybridization region or the 3′ hybridization region of the first padlock probe and the second identifier sequence is complementary to at least a portion of the 5′ hybridization region or the 3′ hybridization region of the second padlock probe;
b) using a first sequencing primer to sequence the first identifier sequence of the first amplicon,
c) removing, cleaving, or blocking the extension product of the first sequencing primer; and
d) using a second sequencing primer different from the first sequencing primer to sequence the second identifier sequence of the second amplicon,
thereby using the sequenced first and second identifier sequences to identify the first RNA analyte and the second RNA analyte.