US 11,866,767 B2
Simultaneous spatio-temporal measurement of gene expression and cellular activity
Cedric Uytingco, Milpitas, CA (US); Layla Katiraee, Castro Valley, CA (US); and Kristen Pham, Pleasanton, CA (US)
Assigned to 10x Genomics, Inc., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Mar. 7, 2023, as Appl. No. 18/179,814.
Application 18/179,814 is a continuation of application No. 17/541,818, filed on Dec. 3, 2021, granted, now 11,624,086.
Application 17/541,818 is a continuation of application No. PCT/US2021/033405, filed on May 20, 2021.
Claims priority of provisional application 63/044,028, filed on Jun. 25, 2020.
Claims priority of provisional application 63/029,121, filed on May 22, 2020.
Prior Publication US 2023/0265489 A1, Aug. 24, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6818 (2018.01); C12N 5/00 (2006.01); C12Q 1/6804 (2018.01); C12Q 1/6837 (2018.01); C12Q 1/6841 (2018.01); C12Q 1/6876 (2018.01); G01N 33/50 (2006.01); G01N 33/543 (2006.01)
CPC C12Q 1/6818 (2013.01) [C12N 5/0062 (2013.01); C12Q 1/6804 (2013.01); C12Q 1/6837 (2013.01); C12Q 1/6841 (2013.01); C12Q 1/6876 (2013.01); G01N 33/5023 (2013.01); G01N 33/5088 (2013.01); G01N 33/54306 (2013.01); C12Q 2600/158 (2013.01); G01N 2458/10 (2013.01)] 30 Claims
 
1. A method for identifying location and/or abundance of an analyte in a plurality of live cells, the method comprising:
(a) contacting the plurality of live cells with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain that binds to a sequence present in the analyte;
(b) perfusing one or more test compounds through a perfusion chamber comprising a gasket and the substrate, wherein the gasket comprises (i) a plurality of apertures, (ii) a plurality of input channels connected to the plurality of apertures, and (iii) a plurality of output channels connected to the plurality of apertures, and wherein the plurality of live cells is located within the perfusion chamber;
(c) hybridizing the analyte to the capture domain of the capture probe;
(d) extending the capture probe using the analyte as a template, thereby generating an extended capture probe;
(e) amplifying the extended capture probe to produce a plurality of extended capture probes; and
(f) sequencing (i) the spatial barcode, or a complement thereof, and (ii) all or part of the sequence of the analyte, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location and/or abundance of the analyte in the plurality of live cells.