	US 11,965,208 B2
	Methods of associating phenotypical data and single cell sequencing data
Christina Chang, San Jose, CA (US); and Sixing Li, San Jose, CA (US)
Assigned to Becton, Dickinson And Company, Franklin Lakes, NJ (US)
Filed by Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed on Apr. 15, 2020, as Appl. No. 16/849,191.
Claims priority of provisional application 62/836,504, filed on Apr. 19, 2019.
Prior Publication US 2020/0332351 A1, Oct. 22, 2020
Int. Cl. C12Q 1/6837 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6858 (2018.01)

CPC C12Q 1/6837 (2013.01) [C12Q 1/6827 (2013.01); C12Q 1/6858 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/16 (2013.01)]

30 Claims

1. A method of labeling a plurality of cells with cell indexing oligonucleotides, comprising:

(a) associating cells in each first partition of a plurality of first partitions with a cellular component-binding reagent of a plurality of cellular component-binding reagents,

wherein each of the cells comprises one or more cellular component targets, wherein the cellular component-binding reagent is associated with a first indexing oligonucleotide with a first cell indexing subsequence, wherein cellular component-binding reagents in each first partition of the plurality of partitions are associated with first indexing oligonucleotides with the same first cell indexing subsequences, and the first cell indexing subsequences are different between at least two first partitions, and wherein the cellular component-binding reagent is capable of specifically binding to the one or more cellular component targets,

thereby generating cells associated with the first cell indexing subsequence, in each first partition of the plurality of first partitions, via the cellular component-binding reagent associated with the first indexing oligonucleotide with the first cell indexing subsequence, wherein the cells in at least two first partitions of the plurality of first partitions are associated with first cell indexing subsequences that are different between the at least two first partitions;

(b) pooling the cells associated with the cellular component-binding reagents in the plurality of first partitions;

(d) associating the distributed cells in each second partition of the plurality of second partitions with a second indexing oligonucleotide of a plurality of second indexing oligonucleotides with a second cell indexing subsequence, wherein the second indexing oligonucleotides of the plurality of second indexing oligonucleotides in at least two second partitions of the plurality of second partitions comprise second cell indexing subsequences that are different between the at least two second partitions,

thereby generating cells associated with the second cell indexing subsequence, in each second partition of the plurality of second partitions, wherein the cells in two second partitions of the plurality of second partitions are associated with different second cell indexing subsequences;

(e) generating cells associated with the cellular component-binding reagent in each second partition of the plurality of second partitions associated with a cell indexing oligonucleotide,

wherein a cell indexing sequence of the cell indexing oligonucleotide comprises:

the first cell indexing subsequence; and

the second cell indexing subsequence of the second cell indexing oligonucleotide associated with the second partition of the plurality of second partitions, via the cellular component-binding reagent associated with the first indexing oligonucleotide with the first cell indexing subsequence associated with each of the cells in the second partition;

(f) pooling the cells associated with the cellular component-binding reagents in the plurality of second partitions;

(g) performing steps (c), (d), (e), and (f) n times, wherein n is a positive integer greater than 1,

thereby generating a plurality of cells associated with unique cell indexing oligonucleotides, each unique cell indexing oligonucleotide comprising:

n cell indexing subsequences; and

a target sequence comprising a sequence complementary to a capture sequence of a stochastic label configured to hybridize to the target sequence, wherein the stochastic label comprises a target binding region comprising the capture sequence, wherein the target binding region is configured to hybridize to a nucleic acid target of a cell of the plurality of cells,

wherein different cells of the plurality of cells are associated with cell indexing oligonucleotides comprising different n cell indexing subsequences;

(h) partitioning the plurality of cells associated with the unique cell indexing oligonucleotides to a plurality of third partitions on a substrate, wherein each third partition of the plurality of third partitions is a distinct spatial location on the substrate and comprises at most a single cell from the plurality of cells associated with the unique cell indexing oligonucleotides;

(i) determining a spatial index of the unique cell indexing oligonucleotides in the plurality of third partitions using n pluralities of probes by sequentially hybridizing and imaging the n pluralities of probes annealing to the n cell indexing subsequences, wherein the spatial index identifies a distinct spatial location of each of the plurality of third partitions on the substrate.