US 12,435,363 B1
Materials and methods for spatial transcriptomics
Caroline Julie Gallant, Stockholm (SE); Augusto Manuel Tentori, Pleasanton, CA (US); Hanyoup Kim, Pleasanton, CA (US); and Rajiv Bharadwaj, Pleasanton, CA (US)
Assigned to 10x Genomics, Inc., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Jun. 9, 2021, as Appl. No. 17/343,271.
Claims priority of provisional application 63/037,189, filed on Jun. 10, 2020.
Claims priority of provisional application 63/037,305, filed on Jun. 10, 2020.
Int. Cl. C12Q 1/6837 (2018.01); G01N 21/64 (2006.01)
CPC C12Q 1/6837 (2013.01) [G01N 21/6428 (2013.01); G01N 21/6458 (2013.01); G01N 2021/6439 (2013.01)] 17 Claims
OG exemplary drawing
 
1. A method of detecting completion of a template switching oligonucleotide (TSO) reaction, the method comprising:
(a) contacting (i) a tissue section with (ii) a plurality of capture probes attached to an array, wherein a capture probe of the plurality comprises a capture domain, wherein the capture domain is positioned at a 3′ end of the capture probe;
(b) releasing one or more target nucleic acids from the tissue section, wherein a target nucleic acid released from the tissue section is bound by the capture domain of the capture probe;
(c) extending the 3′ end of the capture probe using the bound target nucleic acid as a template thereby creating an extended capture probe comprising a sequence that is complementary to the bound target nucleic acid;
(d) adding to the 3′ end of the extended capture probe a first homopolynucleotide sequence;
(e) generating a second strand that comprises in a 5′ to a 3′ direction: a conjugated first agent, a second homopolynucleotide sequence, and a sequence corresponding to a portion of the bound target nucleic acid, wherein the first homopolynucleotide sequence hybridizes to the second homopolynucleotide sequence, and the step of generating the second strand comprises the use of a partially-double stranded template switching oligonucleotide sensor comprising:
(i) a first oligonucleotide comprising a conjugated second agent at its 3′ end, and
(ii) a second oligonucleotide comprising the conjugated first agent at its 5′ end and the second homopolynucleotide sequence at its 3′ end, wherein the second oligonucleotide has a single-stranded overhang at its 3′ end, and wherein:
the conjugated first agent is a fluorophore and the conjugated second agent is a fluorescence quencher; or
the conjugated first agent is a fluorescence quencher and the conjugated second agent is a fluorophore; or
the conjugated first agent is a donor fluorophore and the conjugated second agent is an acceptor fluorophore; or
the conjugated first agent is an acceptor fluorophore and the conjugated second agent is a donor fluorophore; and
(f) measuring fluorescence upon release of the first oligonucleotide from the second oligonucleotide by extension of a 3′ end of the extended first capture probe having the first homopolynucleotide sequence using the second oligonucleotide as a template, thereby detecting the completion of the TSO reaction.