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.
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