US 11,788,120 B2
RNA printing and sequencing devices, methods, and systems
Peter A. Sims, Ardsley, NY (US); Sayantan Bose, Collegeville, PA (US); and Jinzhou Yuan, Edgewater, NJ (US)
Assigned to The Trustees of Columbia University in the City of New York, New York, NY (US)
Appl. No. 16/766,928
Filed by The Trustees of Columbia University in the City of New York, New York, NY (US)
PCT Filed Nov. 27, 2018, PCT No. PCT/US2018/062650
§ 371(c)(1), (2) Date May 26, 2020,
PCT Pub. No. WO2019/104337, PCT Pub. Date May 31, 2019.
Claims priority of provisional application 62/590,889, filed on Nov. 27, 2017.
Prior Publication US 2021/0254143 A1, Aug. 19, 2021
Int. Cl. C12Q 1/6834 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6869 (2018.01); B01L 3/00 (2006.01)
CPC C12Q 1/6834 (2013.01) [B01L 3/5027 (2013.01); B01L 2300/0829 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6869 (2013.01)] 12 Claims
 
1. A method for demultiplexing capture bead barcodes using sequential fluorescence hybridization, wherein said method comprises:
(a) introducing into a plurality of microwells at least one cell and one capture bead, wherein the microwells are formed from a first substrate that faces a second substrate, wherein the capture bead comprises a plurality of oligonucleotide sequences attached thereto, and wherein each oligonucleotide sequence comprises:
i) a PCR handle identical in each oligonucleotide sequence on each capture bead,
ii) a barcode configured for identification of the attached capture bead and an associated cell, wherein the barcode comprises N blocks, wherein each block is an oligonucleotide sequence of length 8 nt to 30 nt chosen from one of N unique sets of M oligonucleotide sequences, wherein N is at least 2 and M is at least 30, wherein said barcode is identifiable by sequential fluorescence hybridization;
iii) a unique molecular identifier (UMI) of length 6 to 16 nucleotides (nt); and
iv) an oligo(dT);
(b) adding a lysis buffer to the microwells, capturing RNA from said at least one cell onto the capture bead, and reverse transcribing; and
(c) optically demultiplexing the capture bead barcodes using sequential fluorescence hybridization, wherein the sequential fluorescence hybridization comprises sequentially introducing mixtures of fluorescently labeled oligonucleotide probes into the microwells, wherein:
i) each of said probes is complementary to one of the oligonucleotide sequences in the unique sets of M oligonucleotide sequences,
ii) said mixtures each comprise at least two probes which are complementary to oligonucleotides sequences within the same unique set of M oligonucleotide sequences, and
iii) the fluorescent label for each of said at least two probes complementary to oligonucleotide sequences within the same unique set of M oligonucleotide sequences is a universal fluorescently labeled oligonucleotide hybridized to a universal sequence included in said probes,
wherein a direct association is provided between phenotypic imaging information acquired from the single cells while located within the microwell and sequence data acquired from the captured and reverse transcribed cellular mRNA.