Patent ID: 11965208
Assignee: BECTON, DICKINSON AND COMPANY
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 4:
5. A method of generating a spatial index, comprising the steps of:
partitioning a plurality of synthetic particles comprising stochastic labels that comprise unique cell indexing oligonucleotides to a plurality of partitions in a substrate, wherein each partition of the plurality of partitions is a distinct spatial location on the substrate and comprises a unique cell indexing oligonucleotide,
wherein the unique cell indexing oligonucleotide comprises a cell indexing sequence comprising a series of n cell indexing subsequences that represent n distinct positions along the cell indexing sequence, wherein n is an integer greater than 1,
wherein each of the plurality of stochastic labels comprises:
a cellular label comprising the cell indexing sequence;
a molecular label, wherein molecular label sequences of at least two stochastic labels of the plurality of stochastic labels comprise different molecule label sequences; and
a target nucleic acid binding region; and

in each partition of the plurality of partitions:
(a) contacting the unique cell indexing oligonucleotide with a plurality of probes,
wherein one or more of the probes of the plurality of probes comprise a detectable moiety, or precursor thereof, wherein at least one of the plurality of probes selectively anneal to cell indexing subsequences with complementary sequences, wherein the at least one probe that selectively anneals to a cell indexing subsequence anneals to at most one of the n distinct positions along the cell indexing sequence;
(b) removing unannealed probes;
(c) imaging the unique cell indexing oligonucleotide, wherein imaging comprises detecting a signal of the detectable moiety at the distinct spatial location on the substrate;
(d) removing the annealed probes and/or the detectable moieties thereof; and
(e) repeating the steps (a)-(d) for a total of n cycles, wherein in each cycle, each probe that selectively anneals to a cell indexing subsequence anneals to a different position of the n distinct positions along the cell indexing sequence than the position of the cell indexing subsequence along the cell indexing sequence to which the at least one probe selectively annealed in any preceding cycle, whereby a spatial index is generated for the unique cell indexing oligonucleotide,

wherein the spatial index comprises a series of: one or more signals; and/or one or more absences of a signal, detected in the n cycles, wherein the sum of the number of the one or more signals and the number of the one or more absences of a signal is n, wherein the spatial index identifies the distinct spatial location on the substrate of each of the plurality of partitions.