Capsule and single-cell combinatorial barcoding

Abstract

Provided herein are methods of combinatorial barcoding of semi-permeable capsules and nucleic acids within encapsulated cells to encode experimental conditions and cell origination.

Description

CAPSULE AND SINGLE-CELL COMBINATORIAL BARCODING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application No.63 / 750,157 filed January 27, 2025, the entire disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is SOMI-007_01WO_SeqList_ST26.xml. The XML file is 1,764,868 bytes, created on January 26, 2026, and is being submitted electronically via USPTO Patent Center.TECHNICAL FIELD

[0003] The present disclosure relates generally to methods of labeling nucleic acid molecules within semi-permeable capsules.BACKGROUND

[0004] Existing approaches to track different experimental conditions for cell culture require either separate labeling or separate processing of the varied experimental conditions. These approaches are costly and time-consuming, limiting the number of experimental conditions that may be tested simultaneously and the cellular throughput. Thus, there is a need for improved high-throughput methods of associating cells with particular cell culture conditions.SUMMARY

[0005] The present disclosure provides combinatorial barcoding methods for barcoding of capsules (e.g., semi-permeable capsules) and at least one nucleic acid molecule in a plurality of nucleic acids comprised in the capsule such that the experimental conditions the capsule was exposed to can be associated with the transcriptomes of the plurality of nucleic acid molecules. In brief, the present disclosure provides methods for labeling the capsule with a trajectory’ label comprised of trajectory barcodes and labeling the capsule and at least one nucleic acid molecule in the plurality of nucleic acids with a capsule label comprised of capsule barcodes. Each trajectory’ barcode corresponds to a specific experimental condition and the order of the trajectory' barcodes in the trajectory’ label corresponds to the order in which the experimental condition occurred. The combination of the capsule barcodes in the capsule label uniquely-1- 330968828identifies the capsule. Because the capsule label is associated with both the trajectory label and the nucleic acids, an association between the experimental conditions the capsule was exposed to and the resulting transcriptome of the cells in the capsule can be made. The present disclosure further provides for methods of resolving the transcriptomes at the single cell level.

[0006] In one aspect, the present disclosure provides a method of labeling a plurality of nucleic acid molecules within a semi -permeable capsule (SPC), comprising:a) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in a trajectory label on the surface of the SPC and a nucleic acid sequence in at least one of the nucleic acids in the plurality of nucleic acids w ithin the SPC; andb) combining the SPC with a second capsule oligonucleotide comprising a second capsule barcode, wherein the second capsule oligonucleotide hybridizes with the first capsule oligonucleotide to produce a capsule label; wherein the capsule label uniquely identifies the SPC; and wherein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label.

[0007] In some embodiments, the nucleic acid sequence in the trajectory label and the nucleic acid sequence in at least one of the nucleic acids is a polyadenylation sequence.

[0008] In one aspect, the present disclosure provides a method of labeling a plurality of nucleic acid molecules within a semi-permeable capsule (SPC), comprising:a) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising the first capsule barcode, wherein the first capsule oligonucleotide hybridizes w ith a trajectory label on the surface of the SPC and the second capsule oligonucleotide hybridizes with a poly-adenylation sequence on at least one of the nucleic acids in the plurality of nucleic acids within the SPC; andb) combining the SPC w'ith a plurality of third capsule oligonucleotides comprising a second capsule barcode, wherein the third capsule oligonucleotide hybridizes with the first and the second capsule oligonucleotides to produce a capsule label; wherein the capsule label uniquely identifies the SPC; and wherein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label.

[0009] In some embodiments, the method comprises sequentially combining the SPC with 3, 4, 5, 6 or more capsule oligonucleotides each comprising a capsule barcode. In some embodiments, the method further comprises, prior to step a), sequentially combining the SPC with a first trajectory oligonucleotide and a second trajectory oligonucleotide, wherein the first and the second trajectory oligonucleotides each comprise a trajectory barcode, wherein the330968828SOMI-007 / 01WG - 352949-2034second trajectory oligonucleotide hybridizes to the first trajectory oligonucleotide to produce the trajectory label; and wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed.|0010] In some embodiments, the SPC is combined with 3, 4, 5, 6 or more trajectory oligonucleotides each comprising a trajectory barcode. In some embodiments, the method further comprises combining the SPC with a trajectory terminator oligonucleotide. In some embodiments, the trajectory' terminator oligonucleotide comprises a first capsule barcode or a universal barcode. In some embodiments, the first and second trajectory oligonucleotides are partially double-stranded DNA oligonucleotides comprising a 3’ single-stranded adapter sequence, a double-stranded trajectory' barcode sequence, and a 5’ single-stranded adapter sequence. In some embodiments, the trajectory terminator oligonucleotide is a single-stranded DNA oligonucleotide.

[0011] In one aspect, the present disclosure provides a method of labeling a plurality of nucleic acid molecules within a semi -permeable capsule (SPC), comprising:a) combining the SPC with a first trajectory oligonucleotide and a second trajectory oligonucleotide, wherein the first and the second trajectory oligonucleotides each comprise a trajectory barcode, wherein the second trajectory' oligonucleotide hybridizes with the first trajectory' oligonucleotide to produce a trajectory' label, and wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed; andb) sequentially combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising a second capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in the trajectory label and the second oligonucleotide hybridizes with the first capsule oligonucleotide to produce a capsule label that uniquely identifies the SPC; wherein the SPC comprises the trajectory' label; and wherein the SPC and the plurality of nucleic acid molecules each comprise the capsule label.

[0012] In some embodiments, the nucleic acid sequence in the trajectory label and the nucleic acid sequence in at least one of the nucleic acids is a polyadenylation sequence.

[0013] In one aspect, the present disclosure provides a method of labeling a plurality of nucleic acid molecules within a semi-permeable capsule (SPC), comprising:a) combining the SPC with a first trajectory oligonucleotide and a second trajectory oligonucleotide, wherein the first and the second trajectory' oligonucleotides each comprise a trajectory barcode, wherein the second trajectory oligonucleotide hybridizes with the first3330968828SOMI-007 / 01WG - 352949-2034trajectory oligonucleotide to produce a trajectory label, and wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed; andb) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising the first capsule barcode, wherein the first capsule oligonucleotide hybridizes with the trajectory label on the surface of the SPC and the second capsule oligonucleotide hybridizes with a poly-adenylation sequence on at least one of the nucleic acids in the plurality of nucleic acids within the SPC; andc) combining the SPC with a plurality of third capsule oligonucleotides comprising a second capsule barcode, wherein the third capsule oligonucleotide hybridizes with the first and the second capsule oligonucleotides to produce a capsule label; wherein the capsule label uniquely identifies the SPC; and wherein the SPC and the plurality of nucleic acid molecules each comprise the capsule label.

[0014] In some embodiments, the SPC is combined with 3, 4, 5, 6 or more trajectory oligonucleotides each comprising a trajectory barcode, wherein each trajectory' oligonucleotide hybridizes with the previous trajectory oligonucleotide to extend the trajectory label. In some embodiments, the first and second trajectory oligonucleotides are partially double-stranded DNA oligonucleotides comprising a 3’ single-stranded adapter sequence, a double-stranded trajectory' barcode sequence, and a 5’ single -stranded adapter sequence. In some embodiments, the 5’ single -stranded adapter sequence of the second trajectory oligonucleotide hybridizes with the 3’ single-stranded adapter sequence of the first trajectory’ oligonucleotide to extend the trajectory label.

[0015] In some embodiments, the step (a) further comprises combining the SPC with a trajectory terminator oligonucleotide. In some embodiments, the trajectory terminator oligonucleotide comprises a first capsule barcode or a universal barcode. In some embodiments, the trajectory' terminator oligonucleotide is a single-stranded DNA oligonucleotide.

[0016] In some embodiments, the method further comprises prior to step (b), combining the SPC with a polynucleotide kinase and a ligase to produce a ligated trajectory label. In some embodiments, the method comprises no more than one ligation step, in some embodiments, the each trajectory barcode corresponds to a unique experimental condition.

[0017] In some embodiments, the method is performed using a plurality of SPCs, wherein each SPC within the plurality is exposed to a unique combination of experimental conditions. In some embodiments, the method is performed using a plurality of SPCs divided into multiple4330968828SOMI-007 / 01WO - 352949-2034sub-pluralities, and wherein each sub-plurality of SPCs is exposed to a unique combination of experimental conditions.

[0018] In some embodiments, the method comprises a reverse transcriptase step whereby the first capsule oligonucleotide is attached to at least one of the plurality of nucleic acids using reverse transcriptase primers that comprise the first capsule barcode. In some embodiments, the second capsule oligonucleotide is attached to the first capsule oligonucleotide using hairpin ligation. In some embodiments, the second capsule oligonucleotide is attached to the first capsule oligonucleotide using bridge-linker ligation. In some embodiments, the second capsule oligonucleotide is attached to the first capsule oligonucleotide using a USER enzyme reaction.

[0019] In some embodiments, the method further comprises release of the trajectory label from the SPC. In some embodiments, the first trajectory oligonucleotide and the second trajectory oligonucleotide each further comprise a 3 " adapter sequence and a 5 ’ adapter sequence. In some embodiments, the 3’ adapter sequence in the first trajectory oligonucleotide is complementary’ to the 5’ adapter sequence in the second trajectory'- oligonucleotide.

[0020] In some embodiments, the SPC comprises an anchor oligonucleotide comprising a single-stranded sequence complementary’ to the 5’ adapter sequence of the first trajectory' oligonucleotide.

[0021] In some embodiments, each of the 5’ and 3’ adapter sequences of the first and second trajectory’ oligonucleotides are at least 16 nucleotides in length. In some embodiments, each of the the 5‘ and 3’ adapter sequences of the first and second trajectory' oligonucleotides are between about 16 nucleotides and about 20 nucleotides in length. In some embodiments, each of the the 5’ and 3’ adapter sequences of the first and second trajectory oligonucleotides are 16, 17, 18, 19, or 20 nucleotides in length.

[0022] In some embodiments, the 5' and 3’ adapter sequences of the firstand second trajectory' oligonucleotides each comprise at least 55%, at least 60%, at least 65%, at least 70%, or 75% GC content. In some embodiments, adapter sequences of the first trajectory oligonucleotide and the second trajectory’ oligonucleotide each comprise about 75% GC content.

[0023] In some embodiments, the first capsule oligonucleotide and the second capsule oligonucleotide each further comprise two adapter sequences. In some embodiments, the adapter sequences of the first capsule oligonucleotide and the second capsule oligonucleotide are each at least 6 nucleotides in length. In some embodiments, the first trajectory barcode and second trajectory' barcode are each between about 16 nucleotides and about 20 nucleotides in length. In some embodiments, the first capsule barcode and second capsule barcode are each between about 16 nucleotides and about 20 nucleotides in length.5330968828SOMI-007 / 01WG - 352949-2034

[0024] In some embodiments, the method is performed using a plurality of SPCs each comprising a plurality of nucleic acids. In some embodiments, the method comprises one or more rounds of pooling and splitting the plurality of SPCs into multiple culture vessels.

[0025] In some embodiments, the plurality of nucleic acids are comprised within one or more cells and the method further comprises contacting a plurality of nucleic acids within a single cell with a first cellular oligonucleotide comprising a first cellular barcode, wherein the cellular barcode uniquely identifies the single cell.

[0026] In some embodiments, the method comprises splitting the single cells into separate vessels prior to contacting the plurality of nucleic acids w ithin the single cell with tire first cellular oligonucleotide. In some embodiments, the method comprises attaching the plurality of nucleic acids within the single cell to barcoded beads prior to contacting the plurality of nucleic acids within the single cell with the first cellular oligonucleotide.

[0027] In one aspect, the present disclosure provides a method of labeling a semi -permeable capsule (SPC) and a plurality of nucleic acid molecules within a plurality of single cells, comprising:a) sequentially combining the SPC with a first trajectory oligonucleotide and a second trajectory’ oligonucleotide, wherein the first and the second trajectory’ oligonucleotides each comprise a trajectory barcode, wherein the second trajectory oligonucleotide hybridizes to the first trajectory’ oligonucleotide to produce a trajectory’ label, and wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed; andb) sequentially combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising a second capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in the trajectory’ label and the second oligonucleotide hybridizes w’ith the first capsule oligonucleotide to produce a capsule label that uniquely identifies the SPC; andc) combining a plurality of nucleic acids from a single cell with a first cellular oligonucleotide comprising a first cellular barcode, wherein the cellular barcode uniquely identifies tire single cell; wherein the SPC comprises the trajectory- label; and w’herein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label; and wherein at least one of the plurality of nucleic acid molecules from the single cell comprises the cellular barcode.

[0028] In one aspect, the present disclosure provides a method of identifying or optimizing a cell differentiation protocol comprising:6330968828SOMI-007 / 01WO - 352949-2034(a) exposing a first SPC comprising a first plurality of cells, each comprising a first plurality of nucleic acid molecules to a first set of sequential experimental conditions, (i) wherein the first SPC is labeled with a first trajectory' label comprising one or more trajectory barcodes that identify each experimental condition in the first set of experimental conditions, (ii) wherein the first SPC and the first plurality of nucleic acid molecules comprise a first capsule label that identifies the first SPC; and (iii) wherein the first plurality of nucleic acids comprise a cellular barcode that iden tifies a single cell in the first plurality of cells;(b) exposing a second SPC comprising a second plurality of cells each comprising a second plurality of nucleic acid molecules to a second set of sequential experimental conditions, (i) wherein the second SPC is labeled with a second trajectory label comprising one or more trajectory barcodes that identify each experimental condition in tire second set of experimental conditions; (ii) wherein the second SPC and tire first plurality of nucleic acid molecules comprise a second capsule label that identifies the second SPC; and (iii) wherein the second plurality of nucleic acids comprise a cellular barcode that identifies a single cell in the second plurality of cells;(c) determining gene expression signatures of the first plurality and second plurality of cells; and(d) correlating each gene expression signature with the set of experimental conditions defined by the trajectory barcodes to identify or optimize the differentiation protocol.

[0029] In one aspect, the present disclosure provides a population of semi -permeable capsules (SPCs) comprising a plurality of nucleic acid molecules, wherein each SPC is labeled with a corresponding capsule label to tire nucleic acid molecules therein; wherein each capsule label comprises a first capsule barcode and a second capsule barcode, and wherein the combination thereof uniquely identifies each SPC,

[0030] In one aspect, tire present disclosure provides a population of labeled nucleic acids, wherein each nucleic acid comprises: (i) a capsule label; and (ii) a sequence corresponding to a gene transcript; wherein the capsule label comprises a combination of barcodes that uniquely identify a semi-permeable capsule (SPC) from which the transcript originated.

[0031] In one aspect, the present disclosure provides a kit comprising: (i) one or more trajectory oligonucleotides; and (ii) one or more capsule oligonucleotides. In some embodiments, the kit further comprises one or more cellular barcodes.

[0032] In one aspect, the present disclosure provides a partially double stranded oligonucleotide comprising: a 5’ single stranded adapter sequence that is between about 16 and 25 nucleotides in length and comprises between 50% and 75% GC content; a double stranded 7330968828barcode sequence; and a 3' single strand adapter sequence that is between about 16 and 25 nucleotides in length and comprises between 50% and 75% GC content. In some embodiments, the 5’ and 3’ adapter are the same length. In some embodiments, the 5’ and 3’ adapter are different lengths. In some embodiments, the 5’ and / or 3’ adapter is 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 5’ and / or 3’ adapter comprise 50%, 55%, 60%, 65%, 70%, or 75% GC content.

[0033] In some embodiments, the barcode sequence corresponds to an experimental condition. In some embodiments, the oligonucleotide is a DNA oligonucleotide.

[0034] In one aspect, the present disclosure provides a library of partially double stranded oligonucleotides, -wherein the oligonucleotides are selected from any described herein, and wherein each oligonucleotide in the library has a unique barcode sequence.

[0035] In one aspect, the present disclosure provides a method of labeling a particle, comprising:hybridizing a first oligonucleotide of any described herein to a ssDNA oligonucleotide affixed to the surface of the particle, wherein the 5' single stranded adapter sequence of the first oligonucleotide hybridizes to the ssDNA oligonucleotide; hybridizing a second oligonucleotide of any any described herein to the first oligonucleotide, wherein the 3’ single stranded adapter sequence of the first oligonucleotide hybridizes with the 5’ single stranded adapter sequence of the second oligonucleotide; hybridizing a third oligonucleotide described herein to the second oligonucleotide, wherein the 3‘ single stranded adapter sequence of the second oligonucleotide hybridizes with tire 5’ single stranded adapter sequence of tire third oligonucleotide; phosphorylating available 5’ ends of the hybridized oligonucleotides; and ligating the hybridized oligonucleotides to form a double-stranded nucleic acid label.

[0036] The barcoding methods described herein accelerate development of cell culture protocols (e.g., cell differentiation protocols) as experimental conditions and their impact on gene expression can be assessed in a high-throughput, multiplex fashion.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1A- FIG. 1C provide schematics of exemplary w orkflow s for labeling capsules and nucleic acids therein. FIG. 1A illustrates trajectory barcoding and capsule barcoding to enable capsule-level transcriptome analysis. FIG. IB illustrates trajectory barcoding, capsule barcoding, and single cell barcoding to enable single-cell transcriptomics analyses. FIG. 1C illustrates the two resulting nucleic acid libraries from the workflows illustrated in FIG. 1A and FIG. IB.8330968828

[0038] FIG. 2 provides a diagram of an exemplary workflow for labeling capsules with a trajectory barcode.

[0039] FIG. 3 provides an exemplary workflow for semi-permeable capsules (SPCs) encapsulating live cells, with acrydite-modified single-stranded anchor oligonucleotides. Optionally, the single-stranded anchor oligonucleotides on an SPC may comprise more than one distinct sequence.,

[0040] FIG. 4A-FIG. 4C provide a diagram of an exemplary workflow for labeling the SPC with trajectory barcodes associated with a first condition (FIG. 4A) and a second condition (FIG. 4B), including the experimental setup.

[0041] FIG. 5 provides a diagram of an exemplary workflow for extending the trajectory barcodes through subsequent rounds of labeling and extension. Multiple rounds of labeling and extension can be performed to produce a final trajectory label representing a specific set of conditions. The final extension comprises an oligonucleotide with a 3' poly- sequence overhang. In some aspects, the final extension comprises an alternative overhang sequence that is not a poly- A sequence.

[0042] FIGs. 6A and 6B provide diagrams of an exemplary workflow of attaching a first capsule barcode to the trajectory' barcodes and the mRNA polynucleotides in the SPC. As shown, the SPCs and cells within the SPCs are fixed and permeabilized followed by pooling the SPCs, splitting them into aliquots across a 96-well plate (FIG. 6A). The SPCs and cells comprised therein are combined with a first capsule oligonucleotide that is a reverse transcription primer comprising a barcode sequence (shown as Index(l) in Fig. 6B and referred to as a capsule barcode sequence in the application) and a polydT sequence. A reverse transcription reaction is performed to produce the first-strand cDNA (FIG. 6B). The SPCs are pooled and split for a second time, followed by combination with a second capsule oligonucleotide as shown in the additional figures herein.

[0043] FIG. 7 provides a diagram of an exemplary workflow for attaching a second capsule oligonucleotide, which contains a second capsule barcode in a hairpin structure (Index 2), to the trajectory' label on the SPC and the reverse transcription primer (which comprises the first capsule barcode) on the cDNA within cells. SPCs are then pooled again.

[0044] FIG. 8 provides a diagram of a next step in the exemplary workflow of Fig. 7, for performing second-strand cDNA synthesis on the nucleic acids within the cells, and second-strand synthesis of the trajectory label on the SPC surface.

[0045] FIG. 9 provides a diagram of a next step in the exemplary workflow of Fig. 7 and Fig.8, for releasing the trajectory label from the SPC surface, followed by adapter ligation and PCR 9330968828SOMI-007 / 01WO - 352949-2034to prepare a trajectory library for sequencing. The SPCs are dissolved to release the cells, which are then split into a 96-well plate. In some embodiments, the trajectory labels may be collected from the supernatant after dissolving the SPCs instead releasing them from the SPC surface.

[0046] FIG. 10 provides a diagram of a next step in the exemplary workflow of Figs. 7-9, for performing Tn5 tagmentation to add adapter sequences to the double-stranded cDNA molecules within the cells.

[0047] FIG. 11 provides a diagram of a next step in the exemplary workflow of Figs. 7-10, for lysing the cells and performing an indexed PCR reaction to add a cellular oligonucleotide comprising a first cellular barcode (Index 3) to the double -stranded cDNA molecules and a plate oligonucleotide comprising a second cellular barcode.

[0048] FIG. 12 provides a diagram of the structure of trajectory library sequences and capsule library sequences generated from the workflow shown in Figs. 7-11. The trajectory library sequences comprise a trajectory label and a capsule label. The capsule label can be used to match tire experimental conditions encoded in the trajectory label with the specific SPC. The capsule library sequences comprise a cellular label, identifying the cell of origin, and a capsule label, identifying the SPC of origin, and therefore the experimental conditions.

[0049] FIG. 13 provides a diagram of an alternative exemplary workflow for attaching a second capsule oligonucleotide to the trajectory label on the SPC and to the reverse transcription primer (which comprises the first capsule barcode) on the cDNA within cells. This workflow uses a bridge-linker ligation method. The second capsule oligonucleotide (Index 2) is a bridging or linker oligonucleotide that is partially complementary to the reverse transcription primer and to the trajectory label, allowing it to hybridize to one another for ligation. The SPCs are then pooled and split into another 96-well plate.

[0050] FIG. 14 provides a diagram of a next step in the exemplary workflow of Fig. 13, for ligating a third capsule oligonucleotide containing a barcode sequence (Index 3) to the second capsule oligonucleotide, on both the trajectory label on the SPC and the cellular nucleic acids, using bridge-linker ligation. The SPCs are then pooled and split into another 96-well plate.

[0051] FIG. 15 provides a diagram of a next step in the exemplary workflow of Fig. 13 and Fig. 14, for releasing the trajectory label from the SPC surface, followed by adapter ligation and fill-in and index PCR to prepare a trajectory library for sequencing. The SPCs are dissolved to release the cells, which are then split into a 96-well plate. In some embodiments, the trajectory' labels may be collected from the supernatant after dissolving the SPCs instead releasing them from the SPC surface.10330968828SOMI-007 / 01WG - 352949-2034

[0052] FIG. 16 provides a diagram of a next step in the exemplary workflow of Figs. 13-15, for ligating a fourth capsule oligonucleotide containing a capsule barcode sequence (Index 4) and a biotin modification using bridge-linker ligation. The cells are pooled and split, followed by cell lysis, reverse crosslinking, and binding of the biotinylated cDNA molecules to streptavidin beads. The cDNA then undergoes a template -switch reaction for cDNA amplification, tagmentation, and sequencing.

[0053] FIG. 17 provides a diagram of the structure of trajectory library sequences and capsule library sequences generated from the workflow of Figs. 13-16. The trajectory library sequences comprise a trajectory label and a capsule label. The capsule label can be used to match the experimental conditions encoded in the trajectory label with the specific SPC. The capsule library sequences comprise a cellular label, identifying the cell of origin, and a capsule label, identifying the SPC of origin, and therefore the experimental conditions.

[0054] FIG. 18 provides a diagram of an alternative exemplary' workflow for attaching a first capsule oligonucleotide comprising a first capsule barcode (Index 1) to the trajectory' label on the SPC and to the cDNA within cells. This workflow' uses a U SER ligation method. Following reverse transcription of mRNA and annealing of the first capsule oligonucleotide to the trajectory' label on the SPC, SPCs are pooled and second-strand cDNA synthesis is performed,

[0055] FIG. 19 provides a diagram of a next step in the exemplary workflow of Fig. 18, for performing U SER cleavage on the reverse transcription primers, followed by splitting the SPCs across a 96-well plate.

[0056] FIG. 20 provides a diagram of a next step in the exemplary' workflow of Figs. 18 and 19, for ligating a second capsule oligonucleotide containing a second capsule barcode (Index 2) to the first capsule oligonucleotide, followed by pooling the SPCs, performing Klenow fill and USER reactions, and splitting the SPCs into a 96-well plate,

[0057] FIG. 21 provides a diagram of a next step in the exemplary' workflow' of Figs. 18-20, for releasing the trajectory label from the SPC surface, followed by adapter ligation, fill-in and index PCRto prepare a trajectory' library for sequencing. The SPCs are dissolved to release the cells, which are then split into a 96-well plate. In some embodiments, the trajectory label may be collected from the supernatant after dissolving the SPCs instead releasing them from the SPC surface.

[0058] FIG. 22 provides a diagram of a next step of the exemplary' workflow' of Figs. 18-21, for ligating an additional capsule oligonucleotide containing a capsule barcode sequence (Index(n)) to the capsule label within cells, followed by pooling the cells, performing Klenow fill and USER reactions, and splitting tire cells into a 96-well plate.11330968828SOMI-007 / 01WO - 352949-2034

[0059] FIGs. 23A-23D provide molecular-level diagrams of exemplary trajectory barcodes, features thereof, and an exemplary7method for labeling SPCs with trajectory barcodes. Fig.23A shows the structure of a trajectory' oligonucleotide comprising a 5’ adapter (presented as a 3’ overhang at the reverse complementary (bottom) strand), a barcode, and a 3’ adapter. Fig.23B provides a schematic illustrating hybridization of the first trajectory oligonucleotide to the ssDNA stub oligonucleotide on the surface of a capsule. Fig. 23C illustrates addition of a second trajectory7oligonucleotide via hybridization through the 3 ’ adapter of the first trajectory oligonucleotide and the 5’ adapter (as presented as a 3’overhang at the reverse complementary (bottom) strand) of the second trajectory oligonucleotide. Fig. 23D illustrates addition of additional oligonucleotides (TBC{n+l}) via hybridization of the 3’ adapter of the previous trajectory oligonucleotide TBC{n} and the 5’ adapter (presented as a 3’ overhang at the reverse complementary (bottom) strand) of the TBC{n+l} trajectory oligonucleotide.

[0060] FIG. 24 provides a molecular-level diagram of a workflow for addition of a first capsule oligonucleotide to the trajectory' label on the SPC. In this workflow, the first capsule oligonucleotide (comprising a first capsule barcode, shown as “CBCI barcode”) comprises a modified design that allows bypassing of tire reverse transcription step, lire first capsule oligonucleotide comprises a sequence (shown as “CBC1 oligo terminator”) that hybridizes with the 3’ adapter of the final trajectory' oligonucleotide (TBC{final}), and a sequence that hybridizes with the 5’ adapter of tire second capsule oligonucleotide (shown as “CBC2-compatible sequence”), Tire CBC1 oligo terminator can be replaced with other types of final oligonucleotide, such as Trajectory' terminator oligonucleotide that has a universal PCR handle w ith UMI for trajectory-only amplification, in this figure and others, the number of ticks (or “■sticks”) depicted in the figure does not necessarily correlate to the number of base pairs.

[0061] FIG. 25A- FIG 25B provide molecular-level diagrams of the single 5’ phosphorylation step (FIG. 25A) and the single ligation step (FIG. 25B), which are performed after hybridization of the last trajectory oligonucleotide as well as first capsule oligonucleotide to the trajectory’ label on the SPC. Sites of 5’ phosphorylation in Fig. 25A and sites of ligation in Fig. 25B are indicated by circles, and the insets show' a larger-scale view of the phosphorylation and ligation.

[0062] FIG. 26A-FIG. 26B provide molecular-level diagrams of addition of a Reverse Transcript primer, including a barcode that matches that of the first capsule oligonucleotide, to the cellular nucleic acids. In FIG. 26A, a oligonucleotide comprising a poly-dT sequence and a capsule barcode, shown as “RT barcode”, and a 5’ overhang that is complementary to the second capsule barcode oligonucleotide (shown as “CBC2-compatible sequence”) is 12330968828hybridized to the polyA sequences of cellular mRNAs and reverse transcription is performed. The RT barcode is experimentally matched to the CBC 1 barcode attached to the trajectory label of the SPC that contains the cell, shown in FIG. 24. In FIG. 26B, instead of the poly-dT sequence, a random hexamer binds internally to cellular mRNAs, but the remaining structure is identical with RT barcode and 5’ overhang for CBC2 -compatible hybridization and ligation. The RT barcode and the CBC1 barcode may be identical or may be different, but allow matching between the RT and CBC1 barcodes by sharing the reaction vessel or designating the capsules to the well of matching RT barcode. FIG. 26C shows tire structure of subsequent capsule oligonucleotides (e.g., a second or third capsule oligonucleotide) comprising a 5" adapter, a barcode, and a 3’ adapter (which is presented as a 5’ overhang on tire reverse complementary [bottom] strand). FIG.26D shows ligation of a second capsule oligonucleotide comprising a second capsule barcode (CBC2) to the first capsule oligonucleotide in both the trajectory label (top) and the cellular nucleic acids (bottom), with a poly-dT RT oligonucleotide primer for the cDNA of cellular nucleic acids. FIG. 26E shows ligation of a second capsule oligonucleotide comprising the same second capsule barcode (CBC2) to the first capsule oligonucleotide in both the trajectory label (top) and the cellular nucleic acids (bottom), using a random hexamer RT oligonucleotide primer for the cDNA of cellular nucleic acids. Additional rounds of ligation can be performed to add additional capsule oligonucleotides to both the trajectory' label and the cellular nucleic acids.

[0063] FIG. 27A- FIG. 27B provide diagrams of exemplary’ workflows for profiling singlecell transcriptomes, following release of cells from SPC Is. The first workflow uses a split-pool method to uniquely label single cells (FIG. 27A) and the second w orkflow' uses droplet-based single cell profiling (FIG. 27B).

[0064] FIG. 28 - FIG. 28B provide diagrams of an exemplary' w'orkflow' for adding a third capsule oligonucleotide comprising a third capsule barcode and a unique molecular identifier (shown as “CBC3 +UMI”) to both the trajectory label on the SPC and the cellular nucleic acids. In the figure, poly-dT reverse transcribed cDNA is showm but random hexamer derived cDNA can be labeled by the same workflow (FIG. 26B, FIG. 26E). This workflow is performed within the capsule and is suitable for capsule-level analysis. The third capsule oligonucleotide hybridizes to tire second capsule oligonucleotide and a ligation is performed (FIG.28A). Next, the cells are released by enzymatic reaction, and the trajectory' labels and cellular nucleic acids (which share the same capsule barcode sequences) are processed separately (FIG. 28B).

[0065] FIG. 29A- FIG. 29B provide diagrams of an exemplary w orkflow' for adding a first cellular oligonucleotide comprising a first cellular barcode and a unique molecular identifier 13330968828SOMI-007 / 01WO - 352949-2034(shown as ‘CBC3+UMI ’) to both the trajectory label on the SPC and the cellular nucleic acids after two or three rounds of capsule barcoding. This workflow involves first enzymatically releasing the cells from the SPC after two or three rounds of capsule barcoding (FIG. 29 A).After centrifugation, the supernatant with the trajectory oligonucleotide is separated and ligated with dummy oligonucleotide with the same structure as CBC3*+UMI (CBC3*=constant). The remaining cell pellet is resuspended and split into wells with first cellular oligonucleotides with distinct barcodes for single-cell analysis using a split+pool approach as in FIG. 27A. Tire first cellular oligonucleotide is hybridized to the second capsule oligonucleotide and a ligation is performed. In the figure, poly-dT reverse transcribed cDNA is shown but random hexamer derived cDNA can be labeled using the same workflow' (FIG. 26B, FIG. 26E). In this workflow, the CBC3 on the trajectory label is the same for all trajectory labels from the same SPC, whereas the CBC3 on the cellular nucleic acids is specific for the single cell. When three rounds of capsule barcoding is performed, effectively CBC4 is being added instead of CBC3 (for t 'O rounds of capsule barcoding). The split & pool ligation of cellular oligonucleotides can be extended to two rounds as well (FIG. 27 A), in which case CBC4 and CBC5+UMI barcodes are added to cellular barcodes (FIG. 27A).

[0066] FIG. 30A- FIG. 30D provide diagrams of an exemplary’ w orkflow7for adding the third capsule oligonucleotide comprising a third capsule barcode and a unique molecular identifier (shown as “■CBC2.5+UMI’’) to both the trajectory label on the SPC and the cellular nucleic acids after pool and split. This w'orkflow is suitable for single-cell analysis using a droplet-based approach as in FIG. 27B. The third capsule oligonucleotide hybridizes to the second capsule oligonucleotide on both the SPC and the cellular nucleic acids (FIG. 30A). Next, the cells are released by enzymatic reaction, followed by centrifugation, and the trajectory labels in the supernatant and cellular nucleic acids in the cells (which share the same capsule barcode sequences) are processed separately (FIG. 30B). The trajectory' label is PCR amplified (FIG.30C) using the PCR handle. The permeabilized cells were mixed with thermostable ligase and bridge oligo, encapsulated into droplet emulsion beads with capture sequences where they are lysed, and the cellular nucleic acids are captured by barcoded beads using a bridge oligo that hybridizes to both the third capsule oligonucleotide (CBC2.5 + UMI) and the bead barcode which represent the cellular barcode (FIG. 30D).

[0067] FIG. 31 provides a molecular-level diagram of the structure of the library of nucleic acids released from SPCs, which contains a trajectory' label and a capsule label. This library' is referred to herein as a trajectory library.14330968828

[0068] FIG. 32A- FIG. 32C provide data demonstrating successful amplification of trajectory label DNA from SPCs (FIG. 32A) and transcriptome library7DNA (FIGs. 32B-32C).

[0069] FIGs. 33A-33F provide data demonstrating complexity and distribution of the transcriptome library.

[0070] FIGs. 33A-33B provide the study design and data for an experiment demonstrating association between trajectory’ barcodes and transcriptional state

[0071] FIGs. 34A-34F provide a study design (FIG. 34A) and UMAP plots demonstrating transcriptomic diversity achieved from capsule-level transcriptomics in this experiment (FIGs.34B-34D) as well as demonstration of select trajectory barcodes associated with the capsule transcriptome (FIGs. 34E-34F).

[0072] FIG. 35 provides data showing release of cells from an SPC.

[0073] FIGs. 36A-36G provide data from an experiment in which single-cell barcoding was performed by a two-level pool & split method similar to single-cell track A (as shown in FIG.27A), following trajectory barcoding and capsule barcoding. FIG. 36A provides UMAP visualization of single-cell transcriptomes from capsule-based combinatorial perturbation screening. Each panel highlights cells (black dots) originating from individual capsules containing 5 or more cells, with remaining cells shown in grey. Capsule Barcodes (CBC) are indicated as BC1-BC2 well positions (e.g., " D1-G4"). The spatial clustering of cells from the same capsule demonstrates successful capture and identification of multi-cellular capsule contents, n = 3,556 total cells from 362 unique capsules; 204 capsules contained >5 cells.

[0074] FIG. 36B provides UMAP visualization of transcriptional cluster analysis, in which unsupervised clustering of single-cell transcriptomes reveals seven distinct cell populations (Leiden clustering resolution = 0.15). “All Clusters” shows all clusters with differential grey shadowing; remaining panels show individual cluster highlighting with two marker genes identified by Wilcoxon rank-sum test. Clusters represent diverse cellular states including:Cluster 0 (n== 1,518): GAPDH, TMSB10 - metabolically active cellsCluster 1 (n=681): MEIS2, CDH11 - mesodermal progenitorsCluster 2 (n=471): SOX2, SEMA6 - neural / pluripotent cellsCluster 3 (n=394): FAM 184 A, FOXA2 - endodermal lineageCluster 4 (n==:219): ALPK2, HAS2 - matrix-producing cellsCluster 5 (n=152): RMRP, PRR9 - proliferating cellsCluster 6 (n=T21): CER1, RHOBTB3 - anterior endoderm1330968828SOMI-007 / 01WG - 352949-2034

[0075] FIG. 36C provides UMAP visualization demonstrating linkage between trajectory barcode data and capsule barcode data, lire left panel provides an overview showing two representative capsules with high-confidence TBC assignments, and the center and right panels show individual capsules with their TBC signatures (format: TBC1-TBC2-TBC3-TBC4-TBC5) linked to their CBC identifiers. This linkage enables tracking of cell lineage relationships within multi-cellular capsules. 98 multi-cell capsules (>5 cells) had high-confidence TBC assignments.

[0076] FIGs.36D provides a log-log plot of barcode rank versus UMI count for quality control assessment. Data from both sublibraries are combined. The characteristic "knee" inflection point separates high-quality cells from empty droplets and ambient RNA. Dashed lines indicate the QC filtering threshold: n:== 3,556 cells retained with UMI > 166. This plot demonstrates successful cell capture and appropriate quality control thresholding.

[0077] FIG. 36E provides a histogram show ing the distribution of cells per Capsule Barcode (CBC) after quality control filtering. A total of 362 unique capsules w'ere identified, containing 1 to 62 cells each (mean: 9.8, median: 6). The solid black line indicates the mean; the dashed grey line indicates the median; the dotted grey line marks the multi -cell threshold (>5 cells). Of the 362 capsules, 204 (56.4%) contained 5 or more cells, qualifying as multi-cell capsules suitable for downstream lineage analysis.

[0078] FIG. 36F-36G provide summary figures showing consolidated visualization of capsule-based single-cell analysis. Six representative multi-cell capsules showing CBC identifiers and cell counts (FIG. 36F) and a cluster overview' shows seven identified populations (left); a cluster legend with cell counts and marker genes; and two representative capsules with TBC-CBC linkage information (right) (FIG. 36G).

[0079] FIGs. 37A-37H provide data from an experiment in which single-cell barcoding w'as performed by a droplet-based single cell profiling method according to single-cell track B (as shown in FIG. 27B), following trajectory barcoding and capsule barcoding. FIG.37A provides UMAP visualization of single-cell transcriptomes of an experiment. Each panel highlights cells (black dots) originating from individual capsules containing 5 or more cells, with remaining cells show'll in grey. Capsule Barcodes (CBC) are indicated as CBC3 CBC2 CBC1 well positions (e.g., " C9__F12__D3"). Hie distribution of individual cell transcriptomes from the same capsule represent the cellular heterogeneity of capsules that are captured from the experiment, n = 824 total cells from 231 unique capsules; 49 capsules contained >5 cells.

[0080] FIG. 37B provides UMAP visualization of transcriptional cluster analysis, in which unsupervised clustering of single-cell transcriptomes reveals four distinct cell populations 16330968828(Leiden clustering resolution::::0.15). “All Clusters” plot shows all clusters with differential grey shadowing; remaining panels show individual cluster highlighting with top two marker genes identified by Wilcoxon rank-sum test. Clusters represent:Cluster 0 (n===590): LDHB, SET - 228 capsules representedCluster 1 (n=213): ACTB, CALR - 118 capsules representedCluster 2 (n=11): MALAT1, SFT2D2 - single-capsule outlier clusterCluster 3 (n=10): PEX13, ALDH18A1 - single-capsule outlier cluster

[0081] FIG. 37C provides UMAP visualization of exemplary' distribution of cell transcriptomes from two capsules with trajectory' information demonstrating linkage between trajectory barcode data and capsule barcodes from single cell transcriptomes. Tire left panel provides an overview showing two representative capsules with confirmed TBC assignments, and the center and right panels show individual capsules displaying their TBC signatures ((TBC1 / TBC2 / TBC3 / TBC4 format from dedicated trajectory library') linked to their corresponding CBC identifiers (CBC3 / CBC2 / CBC1).

[0082] FIGs. 37D provides a log -log visualization of cellular barcode quality assessment. X- axis shows barcode rank (ordered by decreasing Umi count), Y-axis shows total UMI count per barcode. The characteristic "knee" shape distinguishes true cells from empty droplets and ambient RNA. Dashed lines indicate the QC threshold (>300 UMI), yielding n=820 cells passing filter from 5,668 total barcodes with detectable UMI.

[0083] FIG. 37E provides a histogram showing the distribution of cells per capsule (CBC) after QC filtering. A total of 231 unique capsule IDs were identified, with cell counts ranging from 1 to 49 per capsule (mean: 3.6, median: 2). Vertical lines indicate mean (solid), median (dashed), and multi-cell threshold (dotted, >5 cells). 49 capsules (21%) contained >5 cells, qualifying as multi-cell capsules suitable for intra-capsule transcriptional analysis.

[0084] FIG. 37F provides a histogram showing the distribution of Capsule Barcode (CBC) associations per bead barcode across all CellBender-called cells (n=5,669, before UMI filtering). Of 3,305 unique bead barcodes with valid CBC assignments, 2,922 (88.4%) were associated with a single CBC (capsule), while 383 (11.6%) were found in multiple capsules (2-7 CBC combinations), suggesting mild overloading. The mild overloading suggests more capacity for cellular profiling with the droplet procedure by increasing the overloading factor. Mean CBC per bead barcode = 1.17.

[0085] FIGs. 37G-37H provide a consolidated visualization of droplet single-cell analysis workflow. Six representative multi-cell capsules showing CBC identifiers and cell counts 17330968828SOMI-007 / 01WO - 352949-2034demonstrate successful multi-cell capture per capsule (FIG. 37G). A cluster overview shows four transcriptionally distinct populations (left); a cluster legend with cell counts and top marker genes for biological interpretation; and two representative capsules with integrated TBC-CBC linkage demonstrating lineage tracking capability (right) (FIG. 37H).

[0086] FIGs.38A-38C provide data showing the effects of adapter sequence GC content. FIG.38A shows the effect of adapter sequence melting temperature on the relative level of the intended product following ligation or amplification. FIG. 38B shows sequencing data demonstrating relative efficiency of two adapters. FIG. 38C provides data showing improved product amplification with a higher-GC adapter sequence.DET ILED DESCRIPTIONDefinitions

[0087] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein, While not explicitly defined below, such terms should be interpreted according to their common meaning.

[0088] Tlie terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0089] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0090] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.18330968828SOMI-007 / 01WG - 352949-2034

[0091] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2ndedition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc,): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)).

[0092] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or”).

[0093] The term "cell" as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source. In some instances, the cell may be cultured in suspension. In some instances, the cell is a mammalian cell. In some instances, the cell is a human cell. In some instances, the cell is a human stem cell. In some instances, tire stem cell is an induced pluripotent stem cell (iPSC), induced paraxial mesoderm progenitor (iPAM) cell, brown adipocyte cell, muscle stem cell, neural stem cell, totipotent stem cell, hematopoietic stem cell, mesenchymal stem cell, embryonic stem cell, oligopotent stem cell, unipotent stem cell, pluripotent stem cell, or multipotent stem cell.

[0094] " Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may?comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.

[0095] As used herein, "expression" or "express" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0096] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA 19330968828SOMI-007 / 01WG - 352949-2034hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In certain embodiments, the polynucleotide comprises and / or encodes a messenger RNA (mRNA), a short hairpin RNA, and / or small hairpin RNA. In one embodiment, the polynucleotide is or encodes an mRNA. In certain embodiments, the polynucleotide is a double-strand (ds) DNA, such as an engineered ds DNA or a ds cDNA synthesized from a single-stranded RNA,

[0097] The term "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0098] As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, tire term "a cell" includes a plurality of cells, including mixtures thereof.

[0099] As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others. " Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like, " Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0100] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments. Tire term "isolated" is also used herein to refer to polypeptides, proteins and / or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In 20330968828other embodiments, the term "isolated" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0101] As used herein, the term "barcode" or "index" refers to an identification sequence that may be used to associate the product of nucleic acid sequencing with information about the nucleic acid, such as a particular sample, cell, cell nuclei, SPC, or experimental condition. A "barcode" sequence may be single-stranded or double-stranded DNA and may be contained within a longer nucleic acid sequence. As used herein, a "barcoded oligonucleotide" refers to an oligonucleotide sequence containing a barcode sequence that enables the oligonucleotide to be distinguished from other barcoded oligonucleotides. As used herein, an "indexed PCR" refers to a PCR reaction in which one or more barcode sequences are added to a nucleic acid molecule during amplification.

[0102] As used herein, the term “SPC” refers to a semi-permeable capsule; also called a “capsule’’.

[0103] As used herein, the term “anchor oligonucleotide” refers to an oligo attached to the surface of the SPC, with a sequence complementary to a first trajectory' oligonucleotide. Semi-permeable capsules

[0104] The present disclosure provides compositions and methods for combinatorial barcoding of semi-permeable capsules (SPCs) and nucleic acids comprised in the SPCs. Various SPC compositions and methods of generating SPCs are known in the art and can be used in the present disclosure. In some aspects of the disclosure, the SPCs comprise a polyethylene glycol (PEG) shell and dextran core. In some embodiments, the SPCs comprise a polyethylene glycol diacrylate (PEGDA) shell and dextran core. In some aspects of the disclosure, the SPCs comprise a poloxamer shell and a liquid core. Poloxamers are commonly known in the art by trade names such as PLURONIC®, KOLLIPHOR®, and SYNPERONIC® In some aspects of the disclosure, the SPCs are hydrodgel SPCs. In some embodiments, the shell comprises a cross-linked polyethylene glycol)-poly(propylene oxide)-poly(ethylene glycol) diacrylate triblock copolymer {see e.g., WO 2024 / 030526, incorporated herein by reference). In some21330968828SOMI-007 / 01WG - 352949-2034embodiments, the SPC is an SPC as described in WO 2024 / 030526. In some embodiments, the SPC is an SPC as described in US 2020 / 0400538, incorporated herein by reference.

[0105] Methods of producing SPCs are known in the art. See US 2020 / 0400538 and Mazutis et al., Nucleic Acids Res 51, e2 (2023), each incorporated herein by reference. In some embodiments, the methods comprise (a) providing a working core solution comprising a plurality of live cells; (b) providing a working shell solution; (c) providing capsule stabilization oil; (d) generating a plurality of SPCs comprising a plurality of live cells in the core.

[0106] In some embodiments, a plurality of single-stranded DNA oligonucleotides with a 5’ acrydite moiety are included in the working shell solution and are cross-linked to the SPC shell. In such embodiments, these single-stranded acrydite-modified DNA oligonucleotides are referred to as "‘anchor oligonucleotides”. In such embodiments, the methods result in a plurality of SPCs comprising a plurality of live cells in the core and plurality of anchor oligonucleotides cross-linked to the SPC shell. See, e.g.. Fig. 3. The anchor oligonucleotides comprise a nucleotide sequence that hybridizes with adapter sequences of the trajectory oligonucleotides described herein and thereby facilitate attachment of barcodes to the SPC, as described herein.

[0107] In some embodiments, the anchor oligonucleotides comprise an anchor sequence and an acrydite moiety at the 5' end. In some aspects, the anchor oligonucleotides comprise an anchor sequence and an acrydite moiety at the 5' end, followed by a cleavable moiety. In some aspects, the cleavable moiety is a photo-cleavable sequence such as iSpPC, 2-nitrobezyl. In some aspects, the cleavable moiety is a nucleic acid sequence recognized by restriction enzymes, glycosylases, or endonucleases. In some embodiments, the anchor sequences on an SPC may comprise more than one distinct anchor sequence (shown as “register”).

[0108] In some embodiments, an SPC comprises multiple anchor oligonucleotides, wherein each anchor oligonucleotide is the same. See Fig. 3, without the optional orthogonal register. In some embodiments, an SPC comprises at least 2 different anchor oligonucleotides with different anchor sequences. In some embodiments, an SPC comprises at least 3, 4, 5, 6 or more different anchor oligonucleotides with different anchor sequences. See Fig. 4a-4c A nonlimiting example of an anchor oligonucleotide sequence is provided by SEQ ID NO: 215.

[0109] In some embodiments, the anchor sequences represent a particular experimental variable. In such embodiments, the anchor sequences can function as a trajectory barcode. In some embodiments, the SPCs comprise a plurality of anchors representing a plurality of experimental variables, wherein the each anchor sequence in the plurality is unique to a particular experimental variable. In some embodiments, the experimental variable is a particular reagent, reagent concentration, temperature, pH, time, or any combination thereof.22330968828In some embodiments, the experimental variable is reagent that can perturb or affect the cell state. In some embodiments, the reagent is a Wnt agonist, a BMP agonist, a growth factor, an inhibitor, a sugar, an amino acid, a vitamin, a salt, a growth medium, or an enzyme. In some embodiments, the reagent is a combination of reagents, such as a cell culture media. Exemplary cell culture media include NeuroMax, Dulbecco's Modified Eagle Medium (DMEM), DMEM F12, Hams-F12, Minimum Essential Medium (MEM), Roswell Park Memorial Institute 1640 Medium (RPMI), Serum-Free Media (SFM), BenchStable Media, Human Plasma-Like Medium, or other cell culture media known in the art. Exemplary cell culture media also include mesoderm media as described in Loh et al. 2016 Cell vol. 166,2 (2016): 451-467; Chai et al. Nature protocols vol. 11,10 (2016): 1833-50; or Kishimoto et al. Nature protocols vol.17,11 (2022): 2699-2719; ectodermal cell media as described in Hackland et al. Stem cell reports vol. 9,4 (2017): 1043-1052; or Gowhcr and Abdelalim. STAR protocols vol. 3,3 101613, 8 Aug. 2022; and endodermal media as described in Loh et al. Cell stem ' cell vol, 14,2 (2014): 237-52; Hogrebe et al. Nature protocols vol. 16,9 (2021): 4109-4143; or Vanslambrouck et al. Nature protocols vol. 18,11 (2023): 3229-3252.Trajectory Barcoding, Trajectory Oligonucleotides, and Trajectory Barcodes|0110] In some embodiments, the present disclosure provides compositions and methods for trajectory barcoding of SPCs. Herein, the term “trajectory barcoding"’ or “condition-trajectory barcoding” refers to the process of sequentially labeling an SPC with trajectory barcodes, wherein the each trajectory barcode represents a unique experimental variable. Tlie trajectory barcoding methods can be combined with additional combinatorial barcoding methods described herein, such as capsule barcoding to provide unique identifiers for SPCs and singlecell barcoding of nucleic acid molecules (e.g., cellular mRNA) within the SPCs in order to associate the experimental trajectory of a particular SPC and the resulting transcriptomes of cells comprised in the SPC.

[0111] Trajectory' barcoding comprises labeling the SPCs with multiple trajectory' barcodes, wherein each trajectory' barcode represents a unique experimental variable. For example, one trajectory barcode sequence may represent a specific concentration of reagent a, and a second, different trajectory' barcode sequence may represent a specific concentration of reagent 0. In some embodiments, a trajectory barcode sequence may represent multiple experimental conditions, for example a particular cell culture media, a combination of reagents, a combination of time and reagent (e.g., Reagent a given on Day 1 of an experimental protocol), etc. The combination of the different trajectory' barcodes provides a history' of the specific23330968828SOMI-007 / 01WG - 352949-2034experimental variables each SPC encountered. Tlie combination of different conditiontrajectory barcodes can be identified by sequencing methods known in the art.

[0112] In some aspects of the disclosure, trajectory barcoding is carried out by sequentially extending the anchor oligonucleotides on the surface of the SPC shell through sequential hybridization of trajectory' oligonucleotides. A “trajectory oligonucleotide” is an oligonucleotide comprising a trajectory barcode and one or more adaptor sequences. A “trajectory barcode” is a short (e.g., less than 50 nucleotides) nucleic acid sequence that identifies a unique experimental condition or combination of conditions. For example, the experimental condition can be a particular reagent, reagent concentration, temperature, pH, time, or any combination thereof. In some embodiments, the experimental variable is reagent that can perturb or affect the cell state. In some embodiments, the reagent is a Wnt agonist, a BMP agonist, a growth factor, an inhibitor, a sugar, an amino acid, a vitamin, a salt, a growth medium, or an enzyme. In some embodiments, the reagent is a combination of reagents, such as a cell culture media. Exemplary' cell culture media include NeuroMax, Dulbecco's Modified Eagle Medium (DMEM), DMEM F12, Hams-F12, Minimum Essential Medium (MEM), Roswell Park Memorial Institute 1640 Medium (RPMI), Serum-Free Media (SFM), BenchStable Media, Human Plasma-Like Medium, or other cell culture media known in the art.

[0113] An “adapter sequence” is a short (e.g., less than 50 nucleotides) single stranded sequence that is capable of hybridizing with an adapter sequence present in a distinct trajectory’ oligonucleotide or capable of hybridizing to an anchor sequence. In some embodiments, the trajectory oligonucleotides comprise a trajectory barcode sequence flanked by two adapter sequences, a 5’ adapter sequence and a 3’ adapter sequence. In some embodiments, the trajectory’ oligonucleotide is a partially double-stranded DNA oligonucleotide, comprising a 5’ single-stranded adapter sequence, a double-stranded trajectory barcode sequence, and a 3’ single-stranded adapter sequence. See Fig. 23A.

[0114] For both the trajectory’ oligonucleotides and the capsule oligonucleotides described herein, reference to a 5’ adapter and a 3’ adapter is made with reference to the top strand of the duplexed oligonucleotides. As shown in Fig. 23A, the trajectory oligonucleotides described herein comprise 2 polynucleotide strands duplexed at the trajectory barcode region. As used herein, the term “5’ adapter” refers to a molecular overhang present on the side of the duplexed oligonucleotide where the 5’ end of the top strand occurs. In Fig. 23A, this is shown as the left side, and the 5’ adapter is a 3’ overhang on the bottom strand. As used herein, the term “3’ adapter” refers to a molecular overhang present on the side of the duplexed oligonucleotide 24330968828where the 3’ end of top strand occurs. In Fig. 23A, this is shown as the right side, and the adapter sequence is a 3’ overhang on the top strand. Note that a “5’ adapter” may create a 3" overhang because the adapter sequence is present on the bottom strand. Fig. 26C illustrates the analogous structure of the capsule oligonucleotides, wherein the 5’ adapter is present at the 5’ end of tire top strand (in Fig. 26C, this is shown as the left side) and the 3 ’ adapter is present at the 5’ end of the bottom strand (in Fig. 26C, this is shown as the right side). Note that an adapter may be on the 3 ’ end of the barcode region but create a 5 ’ overhang because the adapter sequence is present on the bottom strand.

[0115] In some embodiments, the trajectory barcode is less than 50 nucleotides in length. In some embodiments, the trajectory barcode is between 10 and 30 nucleotides in length. In some embodiments, tire trajectory barcode is between 10 and 25 nucleotides in length, 10 and 20 nucleotides in length, or 10 and 15 nucleotides in length. In some embodiments, the trajectory' barcode is between 15 and 30 nucleotides in length, 15 and 25 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the trajectory' barcode is between 20 and 30 nucleotides in length or 25 and 30 nucleotides in length. In some embodiments, the trajectory barcode is between 15 and 25 nucleotides in length. In some embodiments, the trajectory' barcode is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the trajectory' barcode is 15 nucleotides in length. In some embodiments, the trajectory barcode is 16 nucleotides in length. In some embodiments, the trajectory’ barcode is 17 nucleotides in length. In some embodiments, the trajectory barcode is 18 nucleotides in length. In some embodiments, the trajectory' barcode is 19 nucleotides in length. In some embodiments, the trajectory barcode is 20 nucleotides in length.

[0116] In some embodiments, tire adapter sequence is between 10 and 50 nucleotides in length. In some embodiments, the adapter sequence is between 10 and 25 nucleotides in length, 10 and 20 nucleotides in length, or 10 and 15 nucleotides in length. In some embodiments, the adapter sequence is between 15 and 30 nucleotides in length, 15 and 25 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the adapter sequence is between 20 and 30 nucleotides in length or 25 and 30 nucleotides in length. In some embodiments, the adapter sequence is between 15 and 25 nucleotides in length. In some embodiments, the adapter sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the adapter sequence is 18 nucleotides in length. In some embodiments, the adapter sequence is 19 nucleotides in length. In some embodiments, tire adapter sequence is 20 nucleotides in length. In some embodiments, the adapter sequence is 21 nucleotides in length. In some embodiments, the adapter sequence is 22 nucleotides in 25330968828length. In some embodiments, the adapter sequence is 23 nucleotides in length. In some embodiments, tire 5’ and the 3’ adapter sequences are the same length. In some embodiments, the 5’ and 3’ adapter sequences are different lengths.|0117] In some embodiments, the adapter sequences comprise a high GC percentage. In some embodiments, the adapter sequences comprise at least 50% GC content. In some embodiments, the adapter sequences comprise between about 50% and 75% GC content. In some embodiments, the adapter sequences comprise between about 50% and 70% GC content, between about 50% and 65% GC content, between about 50% and 60% GC content, or between about 50% and 55% GC content. In some embodiments, the adapter sequences comprise between about 55% and 75% GC content, between about 55% and 70% GC content, between about 55% and 65% GC content, or between about 55% and 60% GC content. In some embodiments, the adapter sequences comprise between about 60% and 75% GC content, between about 60% and 70% GC content, or between about 60% and 65% GC content. In some embodiments, the adapter sequences comprise between about 65% and 75% GC content or between about 65% and 70% GC content. In some embodiments, the adapter seqences comprise 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% GC content.

[0118] In some embodiments, the methods described herein comprise combining an SPC comprising a single -stranded DNA anchor oligonucleotide with a first trajectory oligonucleotide, wherein the first trajectory oligonucleotide comprises a trajectory barcode, and wherein the first trajectory oligonucleotide hybridizes with the anchor oligonucleotide, wherein the first trajectory oligonucleotide produces a trajectory label. Mon-limiting examples of first trajectory oligonucleotides for use in such embodiments comprise a combination of sequences selected from Combinations 1-96 of Table 2.

[0119] In some embodiments, the methods described herein comprise sequentially combining an SPC comprising a single-stranded DMA oligonucleotide anchor oligonucleotide with a first trajectory' oligonucleotide and a second trajectory’ oligonucleotide, wherein the first and the second trajectory oligonucleotides each comprise a trajectory barcode, wherein the first trajectory oligonucleotide hybridizes with the anchor oligonucleotide, and wherein the second trajectory oligonucleotide hybridizes to the first trajectory oligonucleotide to produce a trajectory’ label. In such embodiments, non-limiting examples of first trajectory oligonucleotides comprise a combination of sequences selected from Combinations 97-192 of Table 2; and non-limiting examples of second trajectory oligonucleotides comprise a combination of sequences selected from Combinations 193-288 of Table 2.26330968828SOMI-007 / 01WO - 352949-2034

[0120] Herein a “trajectory label” is a double stranded oligonucleotide comprising two or more trajectory oligonucleotides that identifies a set of experimental conditions to which an SPC was exposed. In some embodiments, a trajectory label comprises two trajectory oligonucleotides, in some embodiments, a trajectory label comprises 3, 4, 5, 6, 7, or more trajectory oligonucleotides. In some embodiments, tire trajectory label comprises the anchor sequence and two or more trajectory' oligonucleotides,

[0121] In some embodiments, the methods described herein further comprise sequentially combining an SPC comprising a trajectory label with one or more additional trajectory oligonucleotides. In such embodiments, the one or more additional trajectory oligonucleotides each comprise a trajectory barcode, and the n+1 trajectory' oligonucleotide (whererepresents the third, fourth, fifth, sixth, seventh, or more trajectory oligonucleotide) hybridzes to the n trajectory' oligonucleotide (see FIG. 23D). In some embodiments, the methods comprise combining an SPC comprising a trajectory' label that comprises a first trajectory’ oligonucleotide and a second trajectory' oligonucleotide with a third trajectory oligonucleotide, wherein the first, second, and third trajectory oligonucleotides each comprise a trajectory barcode, wherein the third trajectory oligonucleotide hy'bridizes to the second trajectory' oligonucleotide. In such embodiments, non-limiting examples of first trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 97-192 of Table 2; non-limiting examples of second trajectory oligonucleotides comprise a combination of sequences selected from Combinations 289-384 of Table 2; and non-limiting examples of third trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 385-480 of Table 2.

[0122] In some embodiments, the methods comprise combining an SPC comprising a trajectory’ label that comprises a first trajectory' oligonucleotide, a second trajectory' oligonucleotide, and a third trajectory oligonucleotide with a fourth trajectory' oligonucleotide, wherein the first, second, third, and fourth trajectory oligonucleotides each comprise a trajectory' barcode, wherein the fourth trajectory’ oligonucleotide hybridizes to the third trajectory' oligonucleotide. In such embodiments, non-limiting examples of first trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 97-192 of Table 2; non-limiting examples of second trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 289-384 of Table 2; non-limiting examples of third trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 481-576 of Table 2; and non-limiting examples of fourth trajectory oligonucleotides comprise a combination of sequences selected from Combinations 577-672 of Table 2.27330968828SOMI-007 / 01WG - 352949-2034

[0123] In some embodiments, the methods comprise combining an SPC comprising a trajectory label that comprises a first trajectory oligonucleotide, a second trajectory oligonucleotide, a third trajectory oligonucleotide, and a fourth trajectory oligonucleotide with a fifth trajectory oligonucleotide, wherein the first, second, third, fourth, and fifth trajectory oligonucleotides each comprise a trajectory barcode, wherein the fifth trajectory oligonucleotide hybridizes to the fourth trajectory’ oligonucleotide. In such embodiments, nonlimiting examples of first trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 97-192 of Table 2; non-limiting examples of second trajectory oligonucleotides comprise a combination of sequences selected from Combinations 289-384 of Table 2; non-limi ting examples of third trajectory oligonucleotides comprise a combination of sequences selected from Combinations 481-576 of Table 2; non-limiting examples of fourth trajectory’ oligonucleotides comprise a combination of sequences selected from Combinations 673-768 of Table 2: and non-limiting examples of fifth trajectory oligonucleotides compose a combination of sequences selected from Combinations 769-830 of Table 2.

[0124] In some embodiments, after the trajectory barcoding process, the SPCs and nucleic acids comprised therein undergo a capsule barcoding process, described in detail below. In order for the trajectory label to be compatible with the initial stage of the capsule barcoding process, an overhang on the 3’ end of the trajectory label is needed. As described above and shown in Fig. 23A, the “3" end” of the trajectory label refers to the orientation of tire top strand of the oligonucleotide. In some embodiments, the final oligonucleotide added in the trajectory’ barcoding process is a “trajectory’ terminator oligonucleotide”. Herein, a “trajectory' terminator oligonucleotide” is a single-stranded reverse transcriptase (RT) primer that hybridizes to the 3" adapter sequence of the final trajectory oligonucleotide, thereby producing the necessary overhang on the 3’ end of the trajectory’ label (wherein the overhang is a 5’ overhang on the bottom strand of the trajectory label). See Fig. 24. In some embodiments, the trajectory¬ terminator oligonucleotide is referred to as a “first capsule oligonucleotide". In such embodiments, the first capsule oligonucleotide comprises a sequence (shown as “CBC 1 oligo terminator”) that hybridizes with the 3’ adapter of the final trajectory' oligonucleotide (TBC{ final}), and a sequence that hybridizes with the 5’ adapter of the second capsule oligonucleotide (shown as “CBC2-compatible sequence”). In some embodiments, the CBCT oligo terminator can be replaced with other types of final oligonucleotide, such as Trajectory terminator oligonucleotide that has a universal PCR handle with UMI for trajectory-only amplification, in some embodiments, a trajectory terminator oligonucleotide comprises the first capsule barcode (see description below and shown in Fig. 24 as CBC1 barcode). In some 28330968828SOMI-007 / 01WG - 352949-2034embodiments, the trajectory terminator oligonucleotide does not comprise a capsule barcode (and does not correspond to an experimental condition), but instead serves to add the necessary overhang on the 3’ end of the trajectory' label (wherein the overhang is a 5’ overhang on the bottom strand) such that the capsule oligonucleotides can be added during the capsule barcoding process (referred to herein as a “universal PCR handle with UMF’). In some embodiments, the trajectory terminator oligonucleotide composes a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 15 nucleotides in length. In some aspects, the poly-A sequence comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some aspects the poly-A sequence comprises at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some embodiments, the trajectory terminator oligonucleotide does not comprise a poly-A sequence. In some aspects, the overhang on the 3’ end of the trajectory' label (comprising the 5‘ end of the terminator oligonucleotide) comprises an alternative sequence that facilitates subsequent binding of capsule oligonucleotides ( / .<?., a universal barcode).

[0125] Throughout the trajectory barcoding process, the trajectory oligonucelotides remain associated with one another by virtue of the hydrogen bonds present in the hybridized adapter regions, The GC content and length of the adapter sequences increase the hybridization strength of the oligonucleotides such that multiple oligonucleotides can sequentially hybridize and remain so during tire trajectory barcoding process. The final step in the trajectory barcoding process is to perform 5’ phosphorylation on the available 5’ ends of the trajectory’ oligonucleotides followed by a ligation reaction to form the ligated trajectory- label. See Fig.25A. In some embodiments, the 5’ phosphorylation process is performed using a polymucleotide kinase (PNK) or a mammalian polynucleotide kinase / phosphatase, such as a T4 PNK. In some embodiments, the ligation reaction is performed using a T4 DNA ligase. Importantly, the trajectory barcoding methods described herein comprise a single ligation reaction.

[0126] An exemplary' schematic of the full ligated trajectory' label after completion of the trajectory barcoding process is shown in Fig. 25B. As shown after completion of the ligation step, the trajectory label is a double stranded DNA oligonucletiode comprising n \ l trajectory oligonucleotides representing n +7 experimental conditions and a trajectory terminator oligonucleotide. In some embodiments, n is an integer between 1 and 20. In some embodiments, n is an integer between 1 and 14, 1 and 9, or 1 and 4. In some embodiments, n is an integer between 4 and 19, 4 and 14, 4 and 9, 9 and 19, 9 and 14, or 14 and 19. In some29330968828SOMI-007 / 01WG - 352949-2034embodiments, n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0127] In some embodiments, the trajectory barcoding methods provided herein comprise sequentially combining the SPC with a first trajectory oligonucleotide and a second trajectory oligonucleotide, wherein the first and tire second trajectory oligonucleotides each comprise a trajectory' barcode, wherein the second trajectory oligonucleotide hybridizes to the first trajectory' oligonucleotide to produce the trajectory label, wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed. In some embodiments, the trajectory barcoding methods further comprise combining the SPC labeled with the first and second trajectory oligonucleotides with a trajectory' terminator oligonucleotide, wherein the trajectory label comprises the first and second trajectory oligonucleotides and the trajectory terminator oligonucleotide. In some embodiments, the trajectory terminator oligonucleotide comprises a first capsule barcode. In some embodiments, the trajectory' terminator oligonucleotide comprises a universal barcode.

[0128] In some embodiments, at the end of the trajectory barcoding process, SPCs are processed for trajectory sequencing without a capsule barcoding process. This is referred to herein as “trajectory-only barcoding” method. In some embodiments, SPCs are sorted by fluorescence (“sorted SPCs”) and trajectory labels are amplified in bulk. In some embodiments, the trajectory' label sequences from the sorted SPCs are compared to the original, non-sorted sample, to assess enrichment. In such embodiments, non-limiting embodiments of first, second, third, fourth, and fifth traiectoiy oligonucleotides comprise the combinations of sequences described above, except that the final trajectory oligonucleotide comprises a different combination. In trajectory-only barcoding methods using only a first trajectory oligonucleotide, non-limiting examples of first trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 831-926 of Table 3. In trajectory' -only barcoding methods using only a first trajectory oligonucleotide and a second trajectory oligonucleotide, non-limiting examples of second trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 927-1022 of Table 3. In trajectory' -only barcoding methods using only a first trajectory oligonucleotide, a second trajectory oligonucleotide, and a third trajectory' nucleotide, non-limiting examples of third trajectory oligonucleotides comprise a combination of sequences selected from Combinations 1023-1118 of Table 3. In trajectory -only barcoding methods using only a first trajectory oligonucleotide, a second trajectory' oligonucleotide, a third trajectory nucleotide, and a fourth trajectory nucleotide, non-limiting examples of fourth trajectory' oligonucleotides comprise a combination of sequences selected from Combinations 30330968828SOMI-007 / 01WG - 352949-20341119-1214 of **Table 3**. In trajectory -only barcoding methods using a first trajectory oligonucleotide, a second trajectory oligonucleotide, a third trajectory nucleotide, a fourth trajectory' nucleotide, and a fifth trajectory oligonucleotide, non-limiting examples of fifth trajectory oligonucleotides comprise a combination of sequences selected from Combinations 1215-1275 of Table 3.Capsule barcoding, Capsule Oligonucleotides, and Capsule Barcodes

[0129] In some embodiments, the present disclosure provides a method of labeling a plurality of nucleic acid molecules within an SPC, comprising: a) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in a trajectory label on the surface of the SPC and a nucleic acid sequence in at least one of the nucleic acids in the plurality of nucleic acids within the SPC; and b) combining the SPC with a second capsule oligonucleotide comprising a second capsule barcode, wherein the second capsule oligonucleotide hybridizes with the first capsule oligonucleotide to produce a capsule label; wherein the capsule label uniquely identifies the SPC; and wherein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label. This process of simultaneously labeling an SPC and a plurality of nucleic acids comprised within the SPC with the same label is referred to herein as “capsule barcoding"’. Capsule barcoding uniquely labels each SPC and labels at least one nucleic acid molecule in a plurality of nucleic acid molecules comprised in the SPC with the same capsule label. In some embodiments, all or substantially all of the nucleic acid molecules in the plurality are labeled with the capsule label. In some embodiments, the capsule barcoding process described herein adds additional barcodes to a trajectory label present on the SPC. In some embodiments, the nucleic acid molecules are RNA molecules. In some embodiments, the RNA molecules are mRNA molecules, miRNA molecules, rRNA molecules, tRNA molecules, snRNA molecules, and / or piRNA molecules.

[0130] Herein, the term “capsule oligonucleotide” refers to an oligonucleotide comprising a capsule barcode and one or more adapter sequences. The term “capsule barcode” refers to short e.g., less than 50 nucleotides) nucleic acid sequence that is combined with additional capsule barcodes to uniquely identify an SPC. In some embodiments, the capsule oligonucleotide further comprises two adapter sequences. An “adapter sequence,” in the context of a capsule oligonucleotide, is a short e.g., less than 50 nucleotides) single stranded sequence that is capable of hybridizing with an adapter sequence present in a distinct capsule oligonucleotide or capable of hybridizing to the final 3 ’ overhang of the trajectory label. In some embodiments,31330968828SOMI-007 / 01WO - 352949-2034the capsule oligonucleotide comprises a 5’ adapter sequence and a 3’ adapter sequence (wherein the 3’ adapter sequence is presented as a 5’ overhang on the reverse complement strand).

[0131] In some embodiments, the capsule olignuclotides are hairpin oligonucleotides comprising a 5’ overhang sequence. In some embodiments, the capsule oligonucleotides are double-stranded molecules comprising a 5' overhang sequence (a 5’ adapter sequence) and / or a 3' overhang sequence (a 3’ adapter sequence). In some embodiments, the capsule oligonucleotides are partially double stranded oligonucleotides and comprise a 5’ singlestranded adapter sequence, a double-stranded capsule barcode, and a 3’ single-stranded adapter sequence. See FIG. 26B and CBC2 in Fig. 26C-D. In some embodiments, the capsule barcode is less than 50 nucleotides in length. In some embodiments, the capsule barcode is between 5 and 30 nucleotides in length. In some embodiments, the capsule barcode is between 5 and 25 nucleotides in length, 5 and 20 nucleotides in length, 5 and 15 nucleotides in length, or 5 and 10 nucleotides in length. In some embodiments, the capsule barcode is between 10 and 30 nucleotides in length, 10 and 25 nucleotides in length, 10 and 20 nucleotides in length, or 10 and 15 nucleotides in length. In some embodiments, the capsule barcode is between 15 and 30 nucleotides in length, 15 and 25 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the capsule barcode is between 20 and 30 nucleotides in length or 20 and 25 nucleotides in length. In some embodiments, the capsule barcode is between 25 and 30 nucleotides in length. In some embodiments, the capsule barcode is between 6 and 12 nucleotides in length. In some embodiments, the capsule barcode is between 8 and 10 nucleotides in length. In some embodiments, the capsule barcode is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the capsule barcode is 6 nucleotides in length, In some embodiments, the capsule barcode is 7 nucleotides in length. In some embodiments, the capsule barcode is 8 nucleotides in length. In some embodiments, the capsule barcode is 9 nucleotides in length. In some embodiments, the capsule barcode is 10 nucleotides in length. In some embodiments, the capsule barcode is 11 nucleotides in length. In some embodiments, the capsule barcode is 12 nucleotides in length.

[0132] In some embodiments, the adapter sequence of the capsule oligonucleotide is between 5 and 20 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is between 5 and 15 nucleotides in length, 5 and 10 nucleotides in length, or 10 and 20, 10 and 15 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 5, 6, 7, 8, 9, 10, 11, 12,32330968828SOMI-007 / 01WG - 352949-203413, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 6 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 7 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 8 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 9 nucleotides in length. In some embodiments, the adapter sequence is 10 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 11 nucleotides in length. In some embodiments, the adapter sequence of the capsule oligonucleotide is 12 nucleotides in length. In some embodiments, the 5’ and the 3’ adapter sequences of the capsule oligonucleotide are the same length. In some embodiments, the 5’ and 3’ adapter sequences of the capsule oligonucleotide are different lengths.

[0133] In some embodiments, the methods described herein comprise sequentially combining an SPC comprising a trajectory’ label with a first capsule oligonucleotide and a second capsule oligonucleotide, wherein the first and the second capsule oligonucleotides each comprise a capsule barcode, wherein the first capsule oligonucleotide hybridizes with a 3’ overhang of the trajectory label, and wherein the second capsule oligonucleotide hybridizes to the first capsule oligonucleotide to produce a capsule label. Herein a “capsule label” is a double stranded oligonucleotide comprising two or more capsule oligonucleotides that identifies a unique SPC. In some embodiments, a capsule label comprises two capsule oligonucleotides. In some embodiments, a capsule label comprises 3, 4, 5, 6, 7, or more capsule oligonucleotides.

[0134] Non-limiting examples of first capsule oligonucleotides are provided by SEQ ID NOs: 2-193. A non-limiting example of a trajectory terminator sequence (which hybridizes with the 3 ’ adapter of the final trajectory’ oligonucleotide comprised by the first capsule oligonucleotide) is provided by SEQ ID NO: 1. Non-limiting examples of second and third capsule oligonucleotide sequences are those in the Parse Biosciences v3 kit.

[0135] In some embodiments, the SPCs are pooled and split in between each sequential combination with a capsule oligonucleotide. For example, in some embodiments, the methods comprise pooling a plurality of SPCs, each comprising a trajectory- label, and splitting the plurality into a first set of subpluralities. Each of the first set of subpluralities are then combined with a first capsule oligonucleotide and the first capsule oligonucleotide is ligated to the 3" overhang of the trajectory’ label. The first set of subpluralities are then pooled and split into a second set of subpluralities. Each of the second set of subpluralities are then combined with a second capsule oligonucleotide and the second capsule oligonucleotide is ligated to the 3’ overhang of the first capsule oligonucleotide. In some embodiments, the method further 33330968828SOMI-007 / 01WO - 352949-2034comprises pooling the second set of subpluralities and splitting into a third set of subpluralities. Each of the third set of subpluralities are then combined with a third capsule oligonucleotide and the third capsule oligonucleotide is ligated to the 3’ overhang of the second capsule oligonucleotide. See Fig. 28A.

[0136] At the end of the capsule barcoding process, each SPC comprises a trajectory label and a capsule label molecule and at least one of the nucleic acids comprised in the SPC comprises a capsule label. In some embodiments, all or substantially all of the nucleic acid molecules comprised in the SPC comprise the capsule label. The combination of the trajectory label and capsule label is referred to as a “trajectory library molecule” and the combination of the nucleic acid and the capsule label is referred to as a “transcriptome library molecule”. See Fig. 1C.

[0137] In some embodiments, the SPCs are dissolved and the plurality of trajectory library molecules are collected from the supernatant. In some embodiments, the plurality of trajectory library’ molecules are released from the surface of the SPC. In some embodiments, the plurality of trajectory library molecules are released by enzyme methods. In some embodiments, the enzyme is dextranase. In some embodiments, the enzyme is a restriction enzyme. In some embodiments, the enzyme is a USER enzyme.

[0138] In some embodiments, the SPCs are dissolved after the second capsule oligonucleotide is ligated. In some embodiments, the SPCs are dissolved after the third capsule oligonucleotide is ligated. In embodiments in which the SPCs are dissolved after the second capsule oligonucleotide is ligated, the SPCs are split into a third set of subpluralities after dissolution of the SPC, and the plurality of trajectory library molecules in the supernatant are combined with a third capsule oligonucleotide, which is ligated to the 3 ’ overhang of the second capsule oligonucleotide.

[0139] In some embodiments, the SPCs are dissolved and the plurality' of trajectory’ library¬ molecules are recovered from the supernatant, and the plurality of cells in the SPC are lysed and the plurality of transcriptome library molecules are recovered. Each of the trajectory library molecules and transcriptome library’ molecules are prepared for sequencing. See FIG. 28B.

[0140] In some embodiments, the plurality of trajectory' molecules recovered from the supernatant is cleaned up. The method of clean-up may be any suitable method known in the art. In some embodiments, the plurality of trajectory molecules recovered from the supernatant is cleaned up by magnetic bead (e.g, SPRIselect) cleanup process. Following clean-up, the plurality of trajectory molecules is amplified. In some embodiments, primers specific to both ends of the trajectory library molecules are used for PCR amplification. For example, a first primer specific to a first end of the trajectory’ library molecules and a second primer specific to 34330968828SOMI-007 / 01WG - 352949-2034a second end of the trajectory library molecules are used to amplify the plurality of trajectory library molecules by PCR. In some embodiments, the first primer comprises a first adapter sequence (e.g., a Read1 adapter) and the second primer comprises a second adapter sequence (e.g., a Read2 adapter) for next-generation sequencing, in some embodiments, the PCR primers further include a universal molecular identifier (UMI). Following the amplification, the plurality of trajectory’ library' molecules may be further cleaned up by one or more sizeselection steps. Trajectory library molecules have a specific expected size based on the number of trajectory oligonucleotides and capsule oligonucleotides (e.g., based on the number of rounds of pool + split labeling). In some embodiments, the amplified, cleaned plurality of trajectory library molecules then undergo next-generation sequencing.

[0141] In some embodiments, template-switching oligos (TSOs) are added to the plurality of transcriptome library molecules, followed by second-strand synthesis and cDNA amplification. Non-limiting examples of a TSO sequence include SEQ ID NO: 222. In some embodiments, adapter sequences are added to the plurality of transcriptome library molecules by tagmentation. In some embodiments, adapter sequences are ligated to the plurality of transcriptome library molecules for next-generation sequencing. In some embodiments, the plurality of transcriptome library molecules are amplified by PCR.

[0142] In such embodiments, the resulting transcriptome sequencing information is resolved at the capsule level. In other words, the transcriptome library molecules from all cells present in a particular SPC are sequenced and a capsule-level transcriptome is determined. See Example 7. In some embodiments, the capsule labels identifying the SPC are associated with the corresponding capsule labels on the plurality of nucleic acid sequences, thereby associating the experimental conditions of the SPC with the nucleic acid sequences.Single-Cell Combinatorial Barcoding and Tran scriptome Profiling

[0143] In some embodiments, the present disclosure provides methods for labeling nucleic acids comprised in an SPC for single-cell transcriptome profiling. Tire methods of the present disclosure are compatible with existing combinatorial barcoding approaches for single-cell transcriptome profiling known in the art. Examples of combinatorial barcoding approach known in tire art include SPLiT-seq and sci-RNA-seq3.

[0144] In some embodiments, a plurality of SPCs have undergone the trajectory barcoding and capsule barcoding methods described above. At the end of the capsule barcoding process, each SPC comprises a trajectory' label and a capsule label molecule and at least one of the nucleic acids comprised in the SPC comprises a capsule label. In some embodiments, all or35330968828SOMI-007 / 01WO - 352949-2034substantially all of the nucleic acid molecules comprised in the SPC comprise the capsule label. The combination of the trajectory label and capsule label is referred to as a “trajectory' library molecule” and the combination of the nucleic acid and the capsule label is referred to as a “transcriptome library molecule”. See Fig. 1C.

[0145] Non-limiting embodiments of methods comprising single-cell barcoding and transcriptome profiling are shown in FIGs. 27A and 27B.

[0146] In some embodiments, the SPCs are dissolved and the plurality of trajectory library molecules are recovered from the supernatant, and the plurality of cells in the SPC are split into single cells (e.g., in a plate wherein one cell is present per well). The nucleic acids are then further barcoded with a first cellular barcode to identify nucleic acids from single cells. An additional round of pool and split barcoding can be performed (i.e., wherein the single cells comprising nucleic acids with the first cellular barcode are pooled and split prior to the addition of a second cellular barcode).

[0147] In some embodiments (e.g., in a method comprising steps depicted in FIG. 27A), the method further comprises combining the nucleic acids within a single cell with a first cellular oligonucleotide comprising a first cellular barcode. In some embodiments, the method comprises splitting the single cells into separate vessels prior to contacting the plurality of nucleic acids within a the single cell with a the first cellular oligonucleotide. The separate vessels can be, e.g., wells within a plate, wherein only one cell is present in each well. In some embodiments, the first cellular oligonucleotide comprises a double-stranded barcode sequence, a 5 ’ adapter sequence, and a 3 ’ adapter sequence (wherein the 3 ’ adapter sequence is presented as a 5’ overhang on the reverse complementary strand), in some embodiments, the 5’ adapter sequence hybridizes with the final 3’ adapter of the final capsule oligonucleotide (e.g., the second capsule oligonucleotide). See FIG. 29B.

[0148] Additional rounds of pooling and splitting cells may be performed, for example one additional round or two additional rounds. In some embodiments, the method further comprises combining the nucleic acids within a single cell with a second cellular oligonucleotide comprising a second cellular barcode. In some embodiments, the second cellular oligonucleotide comprises a double -stranded barcode sequence, a 5" adapter sequence, and a 3’ adapter sequence (wherein the 3’ adapter sequence is presented as a 5’ overhang on the reverse complementary strand). In some embodiments, the 5’ adapter sequence hybridizes with the 3’ adapter sequence of the first cellular oligonucleotide.

[0149] In some embodiments (e.g., in a method comprising steps depicted in FIG. 27B), the method further comprises attaching tire plurality of nucleic acids within the single cell to 36330968828SOMI-007 / 01WG - 352949-2034barcoded beads. In some embodiments, the 5' adapter sequence hybridizes with the final capsule oligonucleotide (e.g., the third capsule oligonucleotide CBC2.5), and a ligation is performed. See FIG. 30B. In some embodiments, a droplet-based barcoding method is performed on the plurality of nucleic acids. In some embodiments, the plurality of nucleic acids are captured by barcoded beads using a bridge oligo that hybridizes to both the last capsule oligonucleotide (CBC2.5+UMI) and the bead barcode. A non-limiting example of a bridge oligonucleotide is provided by SEQ ID NO: 214. The three capsule barcodes as well as the the bead barcode comprises the cellular barcode.

[0150] The combination of all cellular oligonucleotides (e.g., the first cellular barcode, or the first and second cellular barcodes) added to the nucleic acids from the single cell produce a cellular label. Herein a ‘"cellular label” is a double stranded oligonucleotide comprising one or more cellular oligonucleotides that identifies a single cell. In some embodiments, a cellular label comprises one cellular oligonucleotide in combination with capsule barcodes. In some embodiments, a cellular label comprises 2, 3, or more cellular oligonucleotides in combination with capsule barcodes.

[0151] At the end of the cellular barcoding process, at least one of the nucleic acids from the single cell comprises a cellular label. In some embodiments, all or substantially all of the nucleic acid molecules from the single cell comprise the cellular lable. The combination of the nucleic acid, tire capsule label, and the cellular label is referred to as a “single-cell transcriptome library’ molecule”.

[0152] In some embodiments, the first cellular oligonucleotide and / or the second cellular oligonucleotide comprises a universal molecular identifier (UMI). In some embodiments, template-switching oligos (TSOs) are added to the plurality of single-cell transcriptome library molecules (from the lysed single cell).

[0153] In some embodiments, the method further comprises second-strand synthesis. In some embodiments, the second-strand synthesis comprises a polymerase reaction to generate double-stranded cDNA molecules comprising the single-cell transcriptome library molecules.

[0154] In some embodiments, the method further comprises an amplification step. In some embodiments, adapter sequences are ligated to the single-cell transcriptome library molecules for next-generation sequencing. In some embodiments, the single-cell transcriptome library molecules are amplified by PCR. In some embodiments, the method further comprises adding a plurality of adapter molecules to the plurality of single-cell transcriptome library molecules. In some embodiments, the method further comprises splitting the plurality of single-cell transcriptome library molecules comprising adapter molecules into two or more separate 37330968828SOMI-007 / 01WG - 352949-2034containers, in some embodiments, the method further comprises providing a plurality of amplification primers to the two or more separate containers, wherein each amplification primer comprises a PCR barcode. In some embodiments, the method further comprises amplifying the plurality of single-cell transcriptome library molecules comprising adapter molecules within the two or more separate containers using the plurality of amplification primers, wherein the amplifying adds the PCR barcode to the plurality of single-cell transcriptome library molecule, and wherein the PCR barcode is unique to each container; and (g) sequencing the single-cell transcriptome library molecules.

[0155] In some embodiments, the plurality of adapter molecules are added using tagmentation. In some embodiments, the plurality of adapter molecules are added using ligation. In some embodiments, the plurality of nucleic acids from the single cell, the single-cell transcriptome library molecules, and / or the double-stranded cDNA molecules are within a plurality of cells. In some embodiments, the plurality of nucleic acids from the single cell, the plurality of singlecell transcriptome library molecules, and / or the double-stranded cDNA molecules are within a plurality of cell nuclei. In some embodiments, the plurality of cells or cell nuclei are fixed and permeabilized prior to providing the first plurality of oligonucleotides to the nucleic acids. In some embodiments, the cells or cell nuclei are fixed using paraformaldehyde. In some embodiments, the cells or cell nuclei are fixed using DSP and methanol. In some embodiments, the cells or cell nuclei are permeabilized using a detergent. In some embodiments, the detergent is Triton X-100. In some embodiments, the fixation and permeabilization preserves the integrity of the cells or cell nuclei and preserves the ability of the SPCs to dissolve. In some embodiments, the PCR barcode identifies a cell or cell nuclei of origin.Compositions and Kits

[0156] In some embodiments, the present disclosure provides an oligonucleotide and libraries thereof, wherein the oligonucleotide is a partially double stranded oligonucleotide comprising: a 5’ single stranded adapter sequence that is between about 16 and 25 nucleotides in length and comprises between 50% and 75% GC content; a double stranded barcode sequence; and a 3’ single strand adapter sequence that is between about 16 and 25 nucleotides in length and comprises between 50% and 75% GC content. In some embodiments, the barcode sequence is a trajectory barcode as described above. In some embodiments, the barcode is a random barcode.

[0157] In some embodiments, the barcode is less than 50 nucleotides in length. In some embodiments, the barcode is between 10 and 30 nucleotides in length. In some embodiments,38330968828the barcode is between 10 and 25 nucleotides in length, 10 and 20 nucleotides in length, or 10 and 15 nucleotides in length. In some embodiments, the barcode is between 15 and 30 nucleotides in length, 15 and 25 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the barcode is between 20 and 30 nucleotides in length or 25 and 30 nucleotides in length. In some embodiments, the barcode is between 15 and 25 nucleotides in length. In some embodiments, the barcode is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0158] In some embodiments, the 5' adapter sequence is between 10 and 50 nucleotides in length. In some embodiments, the 5’ adapter sequence is between 10 and 25 nucleotides in length, 10 and 20 nucleotides in length, or 10 and 15 nucleotides in length. In some embodiments, the 5’ adapter sequence is between 15 and 30 nucleotides in length, 15 and 25 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the 5’ adapter sequence is between 20 and 30 nucleotides in length or 25 and 30 nucleotides in length. In some embodiments, the 5’ adapter sequence is between 15 and 25 nucleotides in length. In some embodiments, the 5’ adapter sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the 5’ adapter sequence is 18 nucleotides in length. In some embodiments, the 5’ adapter sequence is 19 nucleotides in length. In some embodiments, the 5’ adapter sequence is 20 nucleotides in length. In some embodiments, the 5' adapter sequence is 21 nucleotides in length. In some embodiments, the 5’ adapter sequence is 22 nucleotides in length. In some embodiments, the 5’ adapter sequence is 23 nucleotides in length.

[0159] In some embodiments, the 3' adapter sequence is between 10 and 50 nucleotides in length. In some embodiments, the 3’ adapter sequence is between 10 and 25 nucleotides in length, 10 and 20 nucleotides in length, or 10 and 15 nucleotides in length. In some embodiments, the 3’ adapter sequence is between 15 and 30 nucleotides in length, 15 and 25 nucleotides in length, or 15 and 20 nucleotides in length. In some embodiments, the 3’ adapter sequence is between 20 and 30 nucleotides in length or 25 and 30 nucleotides in length. In some embodiments, the 3’ adapter sequence is between 15 and 25 nucleotides in length. In some embodiments, the 3’ adapter sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the 3’ adapter sequence is 18 nucleotides in length. In some embodiments, the 3’ adapter sequence is 19 nucleotides in length. In some embodiments, the 3’ adapter sequence is 20 nucleotides in length. In some embodiments, the 3’ adapter sequence is 21 nucleotides in length. In some39330968828SOMI-007 / 01WO - 352949-2034embodiments, the 3’ adapter sequence is 22 nucleotides in length. In some embodiments, the 3’ adapter sequence is 23 nucleotides in length.

[0160] In some embodiments, the 5’ and the 3’ adapter sequences are the same length. In some embodiments, the 5’ and 3’ adapter sequences are different lengths.

[0161] In some embodiments, the 5’ adapter sequence comprises a high GC percentage. In some embodiments, 5‘ adapter sequence composes at least 50% GC content. In some embodiments, the 5’ adapter sequence comprises between about 50% and 75% GC content. In some embodiments, the 5’ adapter sequence comprises between about 50% and 70% GC content, between about 50% and 65% GC content, between about 50% and 60% GC content, or between about 50% and 55% GC content. In some embodiments, the 5’ adapter sequence comprises between about 55% and 75% GC content, between about 55% and 70% GC content, between about 55% and 65% GC content, or between about 55% and 60% GC content. In some embodiments, the 5’ adapter sequence comprises between about 60% and 75% GC content, between about 60% and 70% GC content, or between about 60% and 65% GC content. In some embodiments, the 5' adapter sequence comprises between about 65% and 75% GC content or between about 65% and 70% GC content. In some embodiments, the 5’ adapter sequence comprises comprise 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% GC content.

[0162] In some embodiments, the 3’ adapter sequence comprises a high GC percentage. In some embodiments, 3‘ adapter sequence composes at least 50% GC content. In some embodiments, the 3’ adapter sequence comprises between about 50% and 75% GC content. In some embodiments, the 3’ adapter sequence comprises between about 50% and 70% GC content, between about 50% and 65% GC content, between about 50% and 60% GC content, or between about 50% and 55% GC content. In some embodiments, the 3‘ adapter sequence comprises between about 55% and 75% GC content, between about 55% and 70% GC content, between about 55% and 65% GC content, or between about 55% and 60% GC content. In some embodiments, the 3’ adapter sequence comprises between about 60% and 75% GC content, between about 60% and 70% GC content, or between about 60% and 65% GC content. In some embodiments, the 3' adapter sequence comprises between about 65% and 75% GC content or between about 65% and 70% GC content. In some embodiments, the 3’ adapter sequence comprises comprise 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% GC content.

[0163] The GC content and length of the adapter sequences in the oligonucleotides described above increase the hybridization strength of the oligonucleotides such that multiple 40330968828oligonucleotides can sequentially hybridize and remain so a barcoding process, in some embodiments, the oligonucleotides described herein enable serial barcoding with 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more oligonucleotides without performing ligation reactions after each oligonucleotide addition. Once the barcoding process is complete, the oligonucleotides are combined with a polynucleotide kinase (PNK) a DNA ligase. The barcoding methods using the oligonucleotides described herein comprise a single ligation reaction.

[0164] In some embodiments, the present disclosure provides libraries of the oligonucleotides described above wherein the library comprises a plurality of oligonucleotides and wherein each oligonucleotide in the plurality comprises a unique barcode sequence.

[0165] In some embodiments, the present disclosure provides a kit comprising: (i) one or more trajectory oligonucleotides; and (ii) one or more capsule oligonucleotides. In some embodiments, the kit further comprises one or more cellular oligonucleotides. The oligonucleotides and barcodes can be provided in separate containers, in multi-well plates, or attached to solid supports such as beads, allowing parallel barcoding of multiple samples or SPCs.

[0166] In some embodiments, the kit further includes one or more primers or adapters comprising sequences compatible with a high-throughput sequencing platform, such as Illumina or nanopore platforms, to enable amplification and sequencing of barcoded nucleic acids. The kit can optionally include enzymes, buffers, and other reagents for nucleic acid amplification, barcoding, and library preparation, together with written instructions describing how to combine the components to generate barcoded sequencing libraries. In some embodiments, the kit further comprises one or more enzymes, such as a polynucleotide kinase (PNK) and / or a DNA ligase.Methods of Use

[0167] In some embodiments, the present disclosure provides a method of labeling a particle with three or more barcodes, comprising: (a) hybridizing a first oligonucleotide to a ssDNA oligonucleotide affixed to the surface of the particle, wherein the first oligonucleotide comprises a 5’ single -stranded adapter sequence, a double stranded barcode sequence and a 3" single stranded adapter sequence (wherein the 3 ’ adapter sequence is presented as a 5 ’ overhang on the reverse complementary strand), wherein the 5’ single stranded adapter sequence of the first oligonucleotide hybridizes to the ssDNA oligonucleotide; (b) hybridizing a second oligonucleotide to the first oligonucleotide, wherein the second oligonucleotide comprises a 5’41330968828SOMI-007 / 01WG - 352949-2034single-stranded adapter sequence, a double stranded barcode sequence and a 3’ single stranded adapter sequence, wherein the 3 ’ single stranded adapter sequence of the first oligonucleotide hybridizes with the 5’ single stranded adapter sequence of the second oligonucleotide; (c) hybridizing a third oligonucleotide to the second oligonucleotide, wherein the third oligonucleotide comprises a 5’ single-stranded adapter sequence, a double stranded barcode sequence and a 3’ single stranded adapter sequence, wherein the 3‘ single stranded adapter sequence of the second oligonucleotide hybridizes with the 5’ single stranded adapter sequence of the third oligonucleotide; (d) phosphorylating available 5" ends of the hybridized oligonucleotides; and (e) ligating the hybridized oligonucleotides to form a double-stranded nucleic acid label.|0168] In some embodiments, the method comprises hybridizing a 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20tholigonucleotide. In some embodiments, 4, 5, 6, 7, 8, 9, or 10 oligonucleotides are hybridized together and ligated with a single ligation step to form a double stranded nucleic acid lable.

[0169] In some embodiments, the particle is selected from the group consisting of semi-permeable capsules, lipid nanoparticles, polymeric nanoparticles, and inorganic or hybrid particles, any of which can be configured to encapsulate or associate with nucleic acids or other cargo.

[0170] In some embodiments, the trajectory barcoding methods and capsule barcoding methods described herein are employed in the development of cellular differentiation protocols. In some embodiments, the present disclosure provides a method of identifying or optimizing a cell differentiation protocol comprising: (a) exposing a first SPC comprising a first plurality of cells, each comprising a first plurality of nucleic acid molecules to a first set of sequential experimental conditions, (i) wherein the first SPC is labeled with a first trajectory label comprising one or more trajectory barcodes that identify each experimental condition in the first set of experimental conditions, (ii) wherein the first SPC and the first plurality of nucleic acid molecules comprise a first capsule label that identifies the first SPC; and (iii) wherein the first plurality of nucleic acids comprise a cellular barcode that identifies a single cell in the first plurality of cells; (b) exposing a second SPC comprising a second plurality of cells each comprising a second plurality of nucleic acid molecules to a second set of sequential experimental conditions, (i) wherein the second SPC is labeled with a second trajectory label comprising one or more trajectory' barcodes that identify each experimental condition in the second set of experimental conditions; (ii) wherein the second SPC and the first plurality of nucleic acid molecules comprise a second capsule label that identifies the second SPC; and (iii)42330968828SOMI-007 / 01WO - 352949-2034wherein the second plurality of nucleic acids comprise a cellular barcode that identifies a single cell in the second plurality of cells; (c) determining gene expression signatures of the first plurality and second plurality of cells; and (d) correlating each gene expression signature with the set of experimental conditions defined by the trajectory barcodes to identify or optimize the differentiation protocol.

[0171] In some embodiments, the cells are stem cells. In some embodiments, the stem cells are embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, or hematopoietic stem cells. In some embodiments, the cells are induced pluripotent stem cells.

[0172] In some embodiments, the methods provided herein enable development of cell differentiation protocols in a high-throughput manner. Using the trajectory and capsule barcoding methods described herein, a multitude of experimental conditions and their combined impact on gene expression can be evaluated in parallel and assessed by high- throughput sequencing methods.EXAMPLES EXAMPLESExample 1: Method overview

[0173] A general overview of exemplary methods is provided by FIG. 1A. in a first part of the workflow (“Trajectory Barcoding”), SPCs encapsulating cells are exposed to series of conditions, and are sequentially labeled with trajectory’ oligonucleotides that combine to form a trajectory label. Tire trajectory’ oligonucleotides each contain barcodes that specifically correspond to each condition (trajectory barcodes).

[0174] In a second part of the workflow (“Capsule Barcoding”), the SPCs and the nucleic acids {e.g., mRNA) within the cells are sequentially labeled with capsule oligonucleotides. The capsule oligonucleotides each contain a capsule barcode, and the combination of capsule barcodes form a capsule label that specifically corresponds to a particular capsule. Within this second part of the workflow, both the cellular nucleic acids (or other modalities, e.g. chromatin etc.) and the SPC undergo the same barcoding steps, such that the capsule label on a particular SPC is the same as the capsule label on the nucleic acids within cells encapsulated by that SPC. A diagram of these shared capsule labels is shown in FIG. IB, in which the circled capsule label comprises three capsule barcode sequences that appear on both a) nucleic acids from cells encapsulated by a particular SPC (left), and b) the trajectory-labeled nucleic acid from that SPC (right).43330968828SOMI-007 / 01WO - 352949-2034

[0175] At this point, the trajectory labels are released from the SPC for trajectory library preparation, and the nucleic acids labeled with the capsule labels are released from the SPCs. As shown in FIG. IB, trajectory' labels released from multiple SPCs form a trajectory library, and cellular nucleic acids released from cells within multiple SPCs form a transcriptome library. Cells can then undergo further round(s) of barcoding to create cell labels made up of cell-level barcodes. Because the capsule labels are shared between trajectory labels and cellular nucleic acids, an association table can be created with single-cell level transcriptome data (or other modalities), profiling the history of conditions to which the cell producing that transcriptome was exposed.Example 2: Generation of semi-permeable capsules comprising trajectory labels.

[0176] This example describes a method for labeling SPCs with a trajectory' label that corresponds to the series of conditions the SPC is exposed to (referred to as “trajectory barcoding”). Live cells are encapsulated in semi-permeable capsules (SPCs) and tire SPCs are then sequentially labeled with trajectory barcodes (FIG. 2). FIG. 3 provides an exemplary workflow for encapsulation of live cells within SPCs by adding the cells to the working core solution. Acrydite-modified single-stranded anchor oligonucleotides with anchor sequences are added to the working shell solution and are crosslinked to the shell of tire SPCs. The anchor sequences can be unique or the same depending on the desired workflow.

[0177] FIGs. 4A-FIG. 4C provide diagrams of an exemplary yvorkfloyv for labeling the SPC with trajectory barcodes associated with experimental conditions utilizing different anchor sequences. A similar process applies yvhen the same anchor sequence is used. SPCs possessing anchor oligos representing agent α and agent β are split into different wells of a 96-well plate. FIG. 4A depicts the first round of trajectory oligonucleotide labeling for a first agent.

[0178] In an exemplary workflow in which multiple anchor oligonucleotides are present on an SPC, a first anchor oligonucleotide may comprise a PCR adapter and a first anchor sequence, yvhereas a second anchor oligonucleotide may comprise a PCR adapter and a second anchor sequence. A first set of trajectory' oligonucleotides, may comprise (i) a first trajectory' oligonucleotide “A” comprising a 5‘ adapter sequence that hybridizes to the first anchor sequence (wherein the 5 " adapter is present as a 3 ’ overhang at the reverse complementary (bottom) strand) a barcode sequence, and a 3’ adapter sequence; and (ii) a first trajectory oligonucleotide B " comprising a 5‘ adapter sequence that hybridizes to the second anchor sequence, a barcode sequence, and a 3’ adapter sequence. A second set of trajectory oligonucleotides, may comprise (i) a second trajectory' oligonucleotide “A” comprising a 5’44330968828SOMI-007 / 01WO - 352949-2034adapter sequence that hybridizes to the 3’ adapter sequence of the first trajectory oligonucleotide “A” (wherein the 5’ adapter is present as a 3’ overhang at the reverse complementary' (bottom) strand), a barcode sequence, and a 3’ adapter sequence; and (ii) a second trajectory oligonucleotide “B” comprising a 5’ adapter sequence that hybridizes to the 3’ adapter sequence of the first trajectory oligonucleotide “B”, a barcode sequence, and a 3’ adapter sequence.

[0179] In the exemplary workflow in which multiple anchor oligonucleotides are present on an SPC, Agent a is added to each well in varying concentrations. A first set of trajectory oligonucleotides is also added to different wells of the 96-well plate. Each trajectory oligonucleotide includes a trajectory barcode sequence specific for a particular concentration of agent a, as well as a complementary sequence to the anchor oligos representing agent a.Trajectory' oligonucleotides are added to each yvell and hybridize to the agent a anchor oligos, thereby recording the concentration of agent a added to each SPC. FIG. 4B depicts the addition of a first set of trajectory oligonucleotides for a second agent. Agent β is added to each well in varying concentrations. A first set of trajectory oligonucleotides (with a barcode sequence specific for each concentration of agent β and a complementary sequence to the anchor oligos representing agent β) is also added to wells of the 96-well plate. The first set of trajectory oligonucleotides are added to each well and hybridize to the agent β anchor oligos, thereby recording the concentration of agent β added to each SPC. Cell culture proceeds following the addition of the first round of agents.

[0180] In a second round of trajectory oligonucleotide labeling, agents α and β are again added to each well in varying concentrations (FIG. 4C). A second set of trajectory oligonucleotides is added to each well. This second set of trajectory oligonucleotides has barcode sequences specific for each concentration of agent α, as well as a complementary sequence to the first set of agent α trajectory oligonucleotides). This second set of trajectory oligonucleotides hybridizes to the first set of agent α trajectory oligonucleotides, thereby recording the second concentration of agent α added to each well and extending the agent α trajectory barcodes. A second set of trajectory oligonucleotides with barcode sequences specific for each concentration of agent β are also added to each well. These oligonucleotides have a complementary sequence to the first set of β trajectory oligonucleotides; they hybridize to the first set of agent β trajectory oligonucleotides, thereby recording the second concentration of agent β added to each well and extending the agent β trajectory barcodes.

[0181] Tlie final trajectory oligonucleotide has a 3' poly-A overhang. In embodiments labeling other modalities, the final oligonucleotide may comprise an overhang with an alternative 45330968828sequence that facilitates binding of subsequent oligonucleotides. In some aspects, the alternative sequence comprises an adapter.

[0182] FIG. 5 provides a diagram of an exemplary workflow for generating the trajectory¬ labels by adding trajectory oligonucleotides (each containing a trajectory barcode) through subsequent rounds of labeling and extension. Trajectory oligonucleotides are added to the trajectory' label by complementary hybridization. Multiple rounds of labeling can be performed to produce a final trajectory label, which comprises a combination of trajectory' barcodes that represent a specific set of conditions. In some aspects, the final trajectory oligonucleotide comprises a 3' poly-A sequence overhang. In some aspects, the final trajectory' oligonucleotide comprises an alternative overhang sequence that is not a poly-A sequence.

[0183] FIGs. 23A-26B provide a molecular-level overview of the method described in this example. An illustrative embodiment of a trajectory' oligonucleotide is provided in FIG. 23A.The trajectory oligonucleotide has a trajectory’ barcode and two flanking adapter sequences. The adapter sequences are unique within a class or "step n" of barcode (BC). For example, a set of first trajectory oligonucleotides may all have a common 5' adapter sequence (wherein the 5’ adapter is present as a 3’ overhang at the reverse complementary' strand) and a common 3' adapter sequence, with variable barcodes. The corresponding set of secondary' trajectory oligonucleotides may all have a common 5' adapter sequence that hybridizes to the common 3’ adapter sequence of the set of first trajectory' oligonucleotides (wherein the 5 ’ adapter is present as a 3’ overhang at the reverse complementary' strand), along with variable barcodes and a common 3' adapter sequence. The 5’ adapter sequence of the first trajectory oligonucleotides differs from the 5’ adapter sequence of the second trajectory oligonucleotides; the same is true for 3’ adapter sequences. This ensures that hybridization of trajectory' oligonucleotides can only occur in sequential order. The trajectory’ oligonucleotides have an extended length to be stable enough for the period of incubation in cell cultures (e.g., at 37°C). The trajectory' barcode has a minimum length of 16 base pairs (bp) with proper melting temperature, and the adapter sequences each have a minimum length of 16bp. The adapters shown in FIG.23 A have adapter sequences of 20bp, and have high GC content to increase stability. The reason for the longer length and higher GC content for the common adapters is to suppress “chimeric” trajectory DNA resulting from random shuffling (e.g., trajectory' oligonucleotides detaching from one SPC and attaching to another within a pool). Specific barcode sequence shuffling is inherently less probable.

[0184] FIG. 23B shows labeling of an SPC with a first trajectory oligonucleotide comprising a trajectory' barcode (shown as “TBC1”). One adapter sequence of the first trajectory 46330968828SOMI-007 / 01WO - 352949-2034oligonucleotide is complementary to the sequence of the single-stranded anchor oligonucleotide (also called, and shown herein as, “stub ssDNA”) on the SPC. Due to the sequence complementarity, an adaptor sequence in the trajectory' oligonucleotide hybridizes with the anchor oligonucleotide. In this way, the length and GC content of the trajectory oligonucleotides allow the SPC to be labeled with a first trajectory barcode without a ligation reaction,

[0185] FIG. 23C shows labeling of an SPC with a second trajectory' oligonucleotide comprising a trajectory barcode (shown as “TBC2”). Similar to the sequence complementarity between the anchor oligonucleotide and the first trajectory oligonucleotide adapter sequence, one adapter sequence of the second trajectory' oligonucleotide is complementary' to the sequence of the second adapter of the first trajectory oligonucleotide. The second trajectory oligonucleotide hybridizes with tire first trajectory oligonucleotide, allowing a second trajectory' barcode to be added without a ligation reaction.

[0186] FIG. 23D shows the sequential labeling of an SPC with a trajectory barcode (shown as TBC{n+l}). Similar to the two previous steps, one adapter sequence of the final trajectory oligonucleotide has a sequence that is complementary to tire sequence of the second adapter of the previous trajectory' oligonucleotide (shown as TBC{n}), At this point, each trajectory oligonucleotide (each comprising a trajectory' barcode) has been added sequentially to the anchor oligonucleotide attached to the SPC without ligation. The combination of trajectory barcodes within the trajectory' label specifically corresponds to the conditions to which the SPC ■was exposed. The final trajectory oligonucleotide has a 3’ overhang sequence of 20bp. Importantly, prior to the ligation step, the trajectory label is held together solely by hydrogen bond interaction between adapters as well as barcodes, without covalent bonding.

[0187] FIG. 24 shows an example of adding a first capsule oligonucleotide to the trajectory label on the SPC. The first capsule oligonucleotide contains a capsule barcode (“CBC1 barcode”), a sequence that is complementary to the sequence of the 3’ overhang of the final trajectory' oligonucleotide (shown as TBC {final}), and a 5’ overhang. The first capsule oligonucleotide hybridizes with the final trajectory oligonucleotide. In this workflow, the first capsule oligonucleotide (comprising a first capsule barcode, shown as “CBC1 barcode”) comprises a modified design that allows bypassing of the reverse transcription step. The first capsule oligonucleotide comprises a sequence (shown as “CBC1 oligo terminator”) that hybridizes with the 3’ adapter of the final trajectory' oligonucleotide (TBC{finaI}), and a sequence that hybridizes with the 5 ’ adapter of the second capsule oligonucleotide (shown as “CBC2-compatible sequence”), lire CBC1 oligo terminator can be replaced with other types 47330968828SOMI-007 / 01WG - 352949-2034of final oligonucleotide, such as Trajectory terminator oligonucleotide that has a universal PCR handle with UMI for trajectory-only amplification.

[0188] After generation of the trajectory label, SPCs are incubated for 30 minutes with T4 Phosphokinase (PNK) to phosphorylate the 5’ end of oligos (FIG. 25A), followed by ligation for 30 minutes at 16°C for nick-sealing (FIG. 25B). Sites of 5" phosphorylation in Fig. 25A and sites of ligation in Fig. 25B are indicated by circles, and the insets show a larger-scale view of the phosphorylation and ligation. The resulting trajectory label will be mostly double¬ stranded with a 5’ single-stranded overhang, compatible with the subsequent workflow' in which capsule barcodes are added sequentially.Example 3: SPC and cell preparation for capsule labeling

[0189] After the addition of the trajectory’ labels to the SPCs as described in Example 1, the SPCs and cells comprised within the SPCs are prepared for the capsule labeling process. The SPCs and cells comprised therein are fixed using paraformaldehyde or DSP and methanol, and permeabilized using Triton X-100 (FIG. 6A). lire fixation and permeabilization conditions are optimized to preserve the integrity of the cells after later release from SPCs and to preserve the ability for the SPCs to dissolve. The fixed and permeabilized capsules with cells can be banked or immediately used in the following steps.

[0190] The fixed / permeabilized SPCs with encapsulated cells are split into separate wells of a 96-well plate. Each well contains reverse transcriptase (RT) primers and reverse transcriptase with appropriate buffers. The RT primers contain a barcode sequence that is unique to each well of the plate (show n here as “Capsule barcode 1”) and a poly-T sequence and / or random hexamers (poly-T sequences, random hexamers, or a combination thereof may be used). FIGs.26A and 26B provide a schematic of an RT primer containing a poly-T sequence (FIG. 26A) or a random hexamer (FIG. 26B) hybridizing to a cellular nucleic acid. A reverse transcription reaction is performed on the mRNA within tire encapsulated cells to generate cDNA with a first capsule barcode on the 3' end of each molecule (wherein the 3’ end refers to the orientation of the cellular nucleic acid strand, and comprises the 5’ end of the reverse complement strand) (FIG. 6B).

[0191] 'Illis results in a 5’ overhang of the first-strand synthesized cDNA. Note that in the current workflow', the trajectory' DNA does not engage with the RT primers, but it will already have the same 5 ’overhang (as shown in FIG. 24) that will be produced by RT. There is 1:1 match of a specific trajectory oligonucleotide adapter sequence of a certain locus (typically the48330968828SOMI-007 / 01WG - 352949-2034last trajectory oligonucleotide) to a given RT well, allowing for matching of the first capsule barcode index of a cDNA molecule to that of a trajectory DNA index.Example 4. Capsule-oligonucleotide labeling within SPCs using hairpin ligation.

[0192] This example describes, after the steps of Example 2, steps for labeling the cDNAs and the SPC-bound trajectory labels with capsule-specific oligonucleotides, using hairpin ligation. The SPCs are pooled back together and split into a 96-well plate containing a second set of capsule oligonucleotides. In this example, tire second capsule oligonucleotides are barcoded hairpin oligos with an overhang with a complementary’ sequence to the 3' end of the RT primers. Each well contains hairpin oligos containing a unique barcode sequence (shown here as “Index 2"’). The hairpin oligos ligate to 5 ’ overhang of both the cDNA and the trajectory’ DNA (on the 3' end of the RT primers on the cDNA molecules within the cells, and on the first capsule oligonucleotides on the SPCs, thereby adding a second capsule oligonucleotide (FIG. 7).

[0193] The SPCs are then pooled again and a second-strand synthesis reaction is performed to generate double-stranded cDNA molecules within the cells and double-stranded oligo sequences on the SPC-bound trajectory’ DNA (FIG. 8),

[0194] At this point, the SPCs can be shunted to a single-cell workflow, or can undergo another round of pool & split capsule labeling (Round 3), at which point it can be shunted to a single¬ cell workflow. Typically after round 3 barcoding, capsule-level transcriptomics with associated trajectory’ is achieved by the subsequent step.

[0195] The SPC-bound trajectory barcode sequences containing the RT and ligation indices are released to the supernatant using an enzyme (e.g., Dexteranase), or restriction enzymes (FIG. 9), or by collecting the sequences from the supernatant after dissolving the SPCs, Standard i7 and i5 adapter sequences are ligated to the 5' and 3' ends of the trajectory barcodes. The anchor sequences on the 5' end of the trajectory barcodes contain a partially complementary^ sequence to the i7 adapter sequence to allow for efficient ligation. A PCR reaction is performed on the trajectory’ barcodes using primers complementary’ for the i7 and i5 adapter sequences.

[0196] The SPCs are dissolved, releasing the cells within the capsules. The cells are split into a 96-well plate and undergo protease digestion and Tn5 tagmentation to add an adapter sequence containing a third index (“Index 3”) to tire cDNA molecules within the cells (FIG.10). The third index sequence is unique to each well. The cells are pooled and split into a 96- well plate, lysed and undergo an indexed PCR to add a fourth index (“Plate index”) to the cDNA molecules (FIG. 11). The labeled transcriptomic library’ (derived from cDNA49330968828SOMI-007 / 01WO - 352949-2034molecules) and the trajectory library (derived from SPC-bound trajectory labels) are sequenced. Indices 1 and 2, present on both the trajectory barcodes and the cDNA molecules, allow the cDNA molecules and experimental conditions to be associated with a particular capsule (FIG. 12). Indices 3 and 4, present on the cDNA molecules, allow the molecules to be associated with a particular cell of origin. Hie cDNA molecules contain cellular barcodes, identifying the cell of origin, and capsule barcodes, identifying the SPC of origin, and therefore the experimental conditions. Associations between the transcriptional state of the cells and the experimental conditions in the SPCs are able to be made.Example 5: Capsule-oligonucleotide labeling within SPCs using bridge ligation.

[0197] This example describes, after the steps of Example 3, steps for labeling the cDNAs and the SPC-bound trajectory’ labels with capsule-specific oligonucleotides, using bridge ligation. The SPCs are pooled back together and split into a 96-well plate containing barcoded oligos annealed to a linker oligo that is complementary to the 3' end of the RT primers and the 5' end of the barcoded oligos. Each well contains barcoded oligos containing a unique barcode sequence. As shown in FIG. 13 (and FIG. 26D, E), the barcoded oligos are ligated to the 3' end of the RT primers on the cDNA molecules within the cells and the trajectory’ barcodes on the SPCs, thereby adding a second index (‘"index 2”). The SPCs are then pooled again and a second-strand synthesis reaction is performed to generate double-stranded cDNA molecules within the cells. The SPCs are split into a 96-well plate containing barcoded oligos annealed to a linker oligo that is complementary to the 3' end of the previous round barcoded oligos and the 5' end of the current round barcoded oligos. Each well contains barcoded oligos containing a unique barcode sequence. As shown in FIG. 14 (and FIG. 28A, 28B), the barcoded oligos are ligated to the 3' end of the previous round barcoded oligos on the cDNA molecules within the cells and the trajectory barcodes on the SPCs, thereby adding a third index (“Index 3”).

[0198] The SPC-bound trajectory barcode sequences containing the RT and ligation indices are released to the supernatant using an enzyme (e.g., Dexteranase), or restriction enzymes (FIG. 15), or by collecting the sequences from the supernatant after dissolving the SPCs. Standard i7 and i5 adapter sequences are ligated to the 5' and 3' ends of the trajectory' barcodes. Hie anchor sequences on the 5’ end of the trajectory barcodes contain a partially complementary’ sequence to the i7 adapter sequence to allow for efficient ligation. A fill-in and PCR reaction is performed on the trajectory barcodes using primers complementary for the i7 and i5 adapter sequences.50330968828SOMI-007 / 01WG - 352949-2034

[0199] The SPCs are dissolved, releasing the cells within the capsules. The cells are split into a 96-well plate and a barcoded oligo containing a fourth index ("‘Index 4”) and a biotin moiety (“B”) is ligated to the cDNA molecules within the cells with a linker oligo that is complementary to the 3' end of the previous round barcoded oligo and to the 5' end of the current round oligo (FIG. 16). The fourth index sequence is unique to each well. The cells are pooled and split into a 96-well plate, lysed, and the cDNA is reverse crosslinked and bound to streptavidin beads. A template-switch reaction is performed with the addition of TSOs, followed by cDNA amplification and tagmentation to add an adapter containing a fifth index. The labeled cDNA molecules and the trajectory barcodes are sequenced. Indices 1, 2, and 3, present on both the trajectory barcodes and the cDNA molecules, allow the cDNA molecules and experimental conditions to be associated with a particular capsule (FIG. 17). Indices 4 and 5, present on the cDNA molecules, allow the molecules to be associated with a particular cell of origin. Associations between the transcriptional state of the cells and the experimental conditions in the SPCs are able to be made.Example 6: Capsule-oligonucleotide labeling within SPCs using USER-enzyme concatenation.

[0200] This example describes, after the steps of Example 3, steps for labeling the cDNAs and the SPC-bound trajectory labels with capsule-specific oligonucleotides, using USER enzyme cleavage. Reverse transcription is performed similarly to that described in Example 2, but slight differences are shown in FIG. 18. The RT primers anneal to the poly- A sequence on the 3' end of the trajectory barcodes, thereby adding a first index (“Index 1”). A second-strand synthesis reaction is performed to generate double-stranded cDNA and to fill in a second strand of the RT primer overhang on the 3' end of the trajectory barcodes. SPCs are pooled and a USER enzyme cleavage is performed, generating an overhang on the RT primers (FIG. 19).

[0201] SPCs are split into a 96-well plate containing barcoded oligos with an overhang on the 5' and 3' ends of the oligos. The barcode sequence is unique to each well. The barcoded oligos are ligated to the RT primers, thereby adding a second index to the cDNA molecules within the cells (“Index 2”) and the trajectory barcodes on the SPCs (FIG. 20). The SPCs are pooled and a Klenow fill reaction and USER reaction are performed. The SPCs are split into a 96-well plate.

[0202] The SPC-bound trajectory barcode sequences containing the RT and ligation indices are released to the supernatant using an enzyme (e.g., Dexteranase), or restriction enzymes (FIG. 21), or by collecting the sequences from the supernatant after dissolving the SPCs.51330968828SOMI-007 / 01WG - 352949-2034Standard i7 and i5 adapter sequences are ligated to the 5' and 3' ends of the trajectory barcodes. The anchor sequences on the 5’ end of the trajectory barcodes contain a partially complementary sequence to the i7 adapter sequence to allow for efficient ligation. A fill-in and PCR reaction is performed on the trajectory barcodes using primers complementary for the i7 and i5 adapter sequences.

[0203] The SPCs are dissolved, releasing the cells within the capsules. The cells are split into a 96-well plate and a barcoded oligo containing a third index is ligated to the cDNA molecules within the cells (FIG.22). The third index sequence is unique to each well. Tire cells are pooled and a Klenow fill and USER reaction is performed. The cells are split into a 96-well plate, lysed and undergo an indexed PCR to add a fourth index to the cDNA molecules. The labeled cDNA molecules and the trajectory barcodes are sequenced. Indices 1 and 2, present on both the trajectory barcodes and the cDNA molecules, allow the cDNA molecules and experimental conditions to be associated with a particular capsule. Indices 3 and 4, present on the cDNA molecules, allow the molecules to be associated with a particular cell of origin. Associations between tire transcriptional state of the cells and the experimental conditions in the SPCs are able to be made.Example 7. Capsule level workflow

[0204] This example describes a workflow for generating capsule-level transcriptomics data. Within the SPC, a final capsule oligonucleotide (for example, a third capsule oligonucleotide) comprising a final capsule barcode (for example, a third capsule barcode) is hybridized to both the trajectory label on the SPC and the cellular nucleic acids (FIG. 28A, third capsule barcode is shown as “CBC3”), Tire final capsule oligonucleotide also comprises a unique molecular identifier (shown as “UMI”). The final capsule oligonucleotide hybridizes to the penultimate capsule oligonucleotide (for example, the third capsule oligonucleotide hybridizes to the second capsule oligonucleotide) and a ligation is performed. Next, the cells are released by enzymatic reaction, and the trajectory labels and cellular nucleic acids (which share the same capsule barcode sequences) are processed separately (FIG. 28B).Example 8. Single-Cell Workflows

[0205] This example describes workflows for single cell transcriptomics analyses.

[0206] FIGS. 27A-27B provide diagrams of two exemplary workflows for profiling single-cell transcriptomes, following release of cells from SPCs,

[0207] In the first of these, referred to as “Track A’’ herein, essentially the same pattern of pool&split barcoding is repeated after the cells are released from tire SPC. FIG. 35 provides 52330968828SOMI-007 / 01WO - 352949-2034data showing release of cells from an SPC. As described above, the SPCs are dissolved and the plurality of trajectory library molecules are recovered from the supernatant. Next, cells in the SPC are split into culture plates with one cell well. The nucleic acids are then further barcoded with a first cellular barcode to identify nucleic acids from single cells. At minimum, if cells are released from capsules after round 2 barcoding, then at least one round of split&pool barcoding is conducted at the single cell level, followed by additional pool&split with either barcoded template-switching oligonucleotides (TSO) or different sample index PCR after library preparation.

[0208] As shown in FIG. 29A, cells were released from SPCs after two rounds of capsule oligonucleotide labeling. In a similar prophetic experiment, cells would be released after a third round of capsule oligonucleotide labeling. Single cells underwent one round of pool+split after release from SPCs. In a similar prophetic experiment, cells would undergo two rounds. After release, the trajectory labels and cellular nucleic acids were separately labeled with a first cellular oligonucleotide comprising a first cellular barcode and a unique molecular identifier (showm as “CBC3+UMI”). The first cellular oligonucleotide was hybridized to the second capsule oligonucleotide and a ligation was performed (FIG. 29A). The CBC3*+ UMI on the trajectory label was the same for all trajectory’ labels from the same SPC, whereas the CBC3 + UMI on the cellular nucleic acids was specific for the single cell.

[0209] In the second single-cell workflow, referred to as ‘Track B” herein, a droplet-based approach is used.

[0210] As shown in FIG. 30A, trajectory labels and cellular nucleic acids were separately labeled after pool&split with the last capsule oligonucleotide comprising the last capsule barcode and a unique molecular identifier (shown as “CBC2.5+UMI+PCR handle+Readl<”). The last capsule oligonucleotide was hybridized to the second capsule oligonucleotide (FIG.30A), then the cells were released by enzymatic reaction. The trajectory labels (in the supernatant) and cellular nucleic acids (in the cellular pellet) share the same capsule barcode sequences and were processed separately (FIG. 30B). Tire trajectory label was PCR amplified (FIG. 30C) using the PCR handle. (FIG. 30D). The cellular nucleic acids underwent droplet-based barcoding. The cellular nucleic acids were captured by barcoded beads using a bridge oligo that hybridizes to both the last capsule oligonucleotide (CBC2.5+UMI) and the bead barcode. Together with the three combinatorial capsule barcodes and the bead barcode comprise the cellular barcode.53330968828SOMI-007 / 01WO - 352949-2034Example 9. Sequencing and computational association of trajectory library sequences with transcriptome library sequences

[0211] FIG. 31 provides a molecular-level diagram of the structure of tire library of nucleic acids released from SPCs (herein called a “trajectory library”, which contains a combination of trajectory oligonucleotides and capsule oligonucleotides.

[0212] Both the trajectory library’ and the tran scriptom ic library’ are paired-end sequenced by next-generation sequencing.

[0213] The trajectory library reads have the following read structure: (Reading from TruSeq Read l->, the actual sequence do not contain the Read 1 sequence) [Trajectory barcode 1]-[adapter sequence 1 (-[Trajectory barcode 2]-[adapter sequence 2]-{... [-[Trajectory barcode m]-[Capsule barcode l]-[Capsule barcode 2]-[Capsule barcode 3 index][UMI] ← (TruSeq 2 read sequence orientation)

[0214] The transcriptome library’ reads have the following read structure: (Reading from TruSeq Read 1 sequence ->)[Tagmented cDNA]-[polydT[Capsule barcode 1 (-[Capsule barcode 2]-[Capsule barcode 3 index] [UMI] ← (TruSeq 2 read sequence)

[0215] The transcriptome library’ sequencing results can be aligned and assembled into a count matrix as any single-cell computational pipelines.

[0216] As for the trajectory library sequencing results, the explicit matching of a certain trajectory' barcode (typically the last trajectory barcode, m) to the RT well defines the first capsule barcode, followed by identical round 2 / 3 indices to match trajectory’ information to a capsule transcriptome.

[0217] In sum, the capsule transcriptome data is obtained with its trajectory (history of conditions encountered) by virtually associating the two sequencing libraries together.Example 10. Amplification of trajectory labels and cellular nucleic acids from SPCs

[0218] SPCs containing cells were treated, trajectory-labeled, and capsule-labeled according to Examples 2-4. Briefly, SPCs were split into RT wells containing uniquely barcoded RT primers (poly-dT and / or random hexamers) and reverse transcriptase with appropriate buffers such that the mRNA of cells within SPCs were reverse transcribed. This resulted in a unique 5’ overhang of the first-strand synthesized cDNA. Note that this workflow, the trajectory label does not engage with the RT primers; instead, the first capsule oligonucleotide adds the same 5 ’overhang that will be produced by RT. There is 1:1 match of a specific trajectory oligonucleotide adapter sequence of a certain locus (typically the last trajectory54330968828oligonucleotide) to a given RT well, allowing for matching of the first capsule barcode index of a cDNA molecule to that of a trajectory DNA index.

[0219] In the next step, SPCs with cells that have the first RT barcoded cDNA as well as the trajectory-labeled DNA were pooled and split again to a second round of wells where each wells contain a 5 ’phosphorylated, overhang barcoded dsDNA, which will ligate to the 5 ’overhang of both the cDNA as well as the trajectory label. This resulted in essentially the same round 2 barcodes.

[0220] Capsules were split into sublibrary fractions, and then for each sublibrary fractions capsules were released by Dexteranase. Released cell aggregates were pelleted. The supernatant (dissolved capsule shell) contains the majority of trajectory DNA. This supernatant was cleaned and followed by the trajectory DNA cleanup and amplification step.

[0221] The supernatant recovered from the dissolution of capsules was cleaned up by a magnetic bead (e.g., SPRIselect) cleanup process, and primers specific to both ends of the trajectory DNA, that typically include adapter sequences for next-generation sequencing are used for PCR amplification, followed by size -selection (as the trajectory DNA has a specific expected size based on the rounds of pool&split barcoding as well as further barcoding for RT well index),

[0222] FIGs. 32A shows specific amplification of trajectory' DNA from SPCs by PCR amplification. The procedure of trajectory barcoding with PNK+ligation generated a durable trajectory' label comprising a combination of trajectory’ barcodes (TBC1, TBC2,...) that can be PCR amplified. Lane 1 represents PCR’ing directly from the SPC-bound trajectory- labels. Lane 2 shows that the supernatant that had capsules does NOT have the trajectory DNA (the DNA is firmly attached to capsules). Lane 3 represents PCR’ing after dissolving the capsules to release the cells, where the dissolved capsule shells will contain the trajectory- DNA which is amplified.

[0223] FIGs. 32B (left) provides Tapestation data with fluorescence probes that display the intensity'- of DNA molecules for a given size of the transcriptome library generated. “Lower”, and ‘Upper” represent the spike-in control, which are known to have specific size to calibrate the length. The graph shows analysis trace post-amplification and bead cleanup of ligated trajectory + capsule transcriptome barcode. FIGs. 32B (right) provides statistics from the Oxford nanopore sequencing result of the library-. The graph shows fragment length distribution colored by match to expected product (trajectory + capsule transcriptome barcode) as assessed by nanopore sequencing. Oxford nanopore sequencing reads the actual DNA sequence from a single molecule, such that its read out represents the composition of the library. The graph 55330968828SOMI-007 / 01WG - 352949-2034shows the histogram of all the read’s sequencing length. It demonstrates a 338bp-length peak, which exactly matches the expected library structure. These data indicate that clear, specific sequence was amplified from the transcriptome library labeled and generated by this method. FIGs. 32C provides data showing successful recovery of capsule-level transcriptome library. Specifically, the graphs show a cDNA library trace for capsule-level transcriptome using split-pool labeling (left) and sequencing library’ trace for capsule-level transcriptome using splitpool labeling (right).

[0224] Next, the complexity and distribution of the trajectory DNAs and the transcriptome library’ were assessed. FIGs. 33A shows a log-log plot of the sequenced library count matrix, as determined by sequencing analysis utilizing Kallisto Bustools-Cellbender. The X axis corresponds to barcodes representing capsule IDs and tlie Y axis corresponds to UMI counts per capsule ID. These data show that the transcriptome library' displays the proper, expected library’ complexity. FIG. 33B shows the extracted barcode sequences from the first capsulebarcode (CBC1) well. Dots represent different sublibraries, and error bars represent technical replicates. These data show that the loaded capsules show the expected range of unique capsule counts, demonstrating that the capsule processing pipeline and library' generation were effective.

[0225] FIGs. 33C and 33D, respectively, show the distribution of the second, and third, capsule barcodes (CBC2, CBC3) from each well of the 96-well plate (left) and the number of capsules with each CBC code (right), The bottom -right four wells in FIG.33C were not loaded, demonstrating minimal ambient RNA contamination These data demonstrate near-uniform distribution from the pool and split procedure for round tw o and three of capsule barcoding according to this method. FIG. 33E shows the aggregate UMI distribution of capsule-level transcriptomes, showing distribution of cell occupancy among capsules. The QI to Q4 represent fitting the capsule distribution based on UMI into four gaussian mixtures. These data are further visualized in FIG. 33F, which provides an image depicting cell occupancy within capsules (right) as they correspond to cell occupancy (position on the X-axis). In this experiment, 42,500 SPCs were found to contain cells, based on the UMI counts per SPC. Example 11. Data supporting Capsule-level transcriptome-to-capsule association

[0226] FIGs. 34A provides the study design for an experiment demonstrating association between trajectory barcodes and transcriptional state. SPCs containing cells were cultured in a range of conditions (shown here as “contexts”: 18 contexts in the first stage, 108 contexts in the second stage, and 648 contexts in the third stage. In all, 74,000 trajectories (series of56330968828SOMI-007 / 01WO - 352949-2034contexts) were used. Transcriptomic information for a five-locus transcriptomic library was overlaid. FIGs. 34B-34E provides UMAP plots demonstrating transcriptomic diversity achieved from capsule-level transcriptomics in this experiment. In the given plots, capsules are annotated by cell type (FIG. 34B), day (FIG. 34C), and lineage (FIG. 34D). Transcriptomic information for an exemplary set of individual barcode loci is overlaid on the final panel (FIG.34E), The distribution of trajectory’ barcodes corresponds to transcriptional status, showing successful association of transcriptional state to trajectory' barcode.Example 12. Data supporting single-cell transcriptome-to-capsule association

[0227] This example describes an experiment in which single-cell barcoding was performed using a two-level pool & split combinatorial barcoding method according to single-cell track A (as shown in FIG. 27A), following trajectory’ barcoding and capsule barcoding.

[0228] Cells within SPCs had undergone combinatorial barcoding rounds 1-2 (described in Example 8), generating Capsule Barcodes (CBC) defined by the combination of BC1-BC2 well positions (e.g., " D1-G4"). Following decapsulation with dextranase, cells were dissociated using TrypLE Express, filtered through a 40-micron strainer, and centrifuged into a pellet. Supernatant was collected, ligated with a small number of first cellular oligonucleotides (CBC3), and followed up for trajectory (TBC) library preparation as described above. The pellet was resuspended into single cell suspension and split into the CBC3 well positions for cell-level barcode ligation. The cells were split into sublibraries, to the point of capsule-level barcode procedure described before..

[0229] Raw sequencing data was processed through a two-stage quality control pipeline: QC1 (Scanpy: cell filtering, normalization, log transformation) followed by QC2 (Seurat: TBC integration). Cells were filtered to retain those with sufficient UMI counts (>166 UMI). Dimensionality reduction was performed using PCA (30 components) followed by UMAP visualization (20 PCs, 15 nearest neighbors, min _dist=0.3). Unsupervised clustering was performed using the Leiden algorithm (resolution=0.15). Gene symbols were converted from Ensembl IDs using the mygene database.

[0230] Data:

[0231] FIG. 36A provides UMAP visualization of single-cell transcriptomes from capsule-based combinatorial perturbation screening. Each panel highlights cells (black dots) originating from individual capsules containing 5 or more cells, with remaining cells shown in grey. Capsule Barcodes (CBC) are indicated as BC1-BC2 well positions (e.g., " D1-G4"). The spatial clustering of cells from the same capsule demonstrates successful capture and identification of57330968828SOMI-007 / 01WG - 352949-2034multi-cellular capsule contents, n = 3,556 total cells from 362 unique capsules; 204 capsules contained >5 cells.

[0232] FIG. 36B provides UMAP visualization of transcriptional cluster analysis, in which unsupervised clustering of single-cell transcriptomes reveals seven distinct cell populations. “All Clusters” shows all seven clusters with differential grey shading. Each population is shown a separate panel, with the individual cluster highlighting with top two marker genes identified by Wilcoxon rank-sum test.

[0233] FIG. 36C provides UMAP visualization demonstrating linkage between trajectory barcode data and capsule barcode data. The left panel provides an overview showing two representative capsules with high-confidence TBC assignments, and the center and right panels show individual capsules with their TBC signatures (format: TBC1-TBC2-TBC3-TBC4-TBC5) linked to their CBC identifiers. This linkage enables tracking of cell lineage relationships within multi-cellular capsules. 98 multi-cell capsules (>5 cells) had high-confidence TBC assignments.

[0234] FIGs.36D provides a log-log plot of barcode rank versus UMI count for quality control assessment. Data from both sublibraries are combined. The characteristic "knee" inflection point separates high-quality cells from empty droplets and ambient RNA. Dashed lines indicate the QC filtering threshold: n = 3,556 cells retained with UMI > 166. This plot demonstrates successful cell capture and appropriate quality control thresholding.

[0235] FIG. 36E provides a histogram showing the distribution of cells per Capsule Barcode (CBC) after quality control filtering. A total of 362 unique capsules 'ere identified, containing 1 to 62 cells each (mean: 9.8, median: 6). The solid black line indicates the mean; the dashed grey line indicates the median; the dotted grey line marks the multi -cell threshold (>5 cells). Of the 362 capsules, 204 (56.4%) contained 5 or more cells, qualifying as multi-cell capsules suitable for downstream lineage analysis.

[0236] FIG. 36F-G provide summary figures showing consolidated visualization of capsule-based single-cell analysis. Six representative multi-cell capsules showing CBC identifiers and cell counts (FIG. 36F) and a cluster overview shows seven identified populations (left); a cluster legend with cell counts and marker genes; and two representative capsules with TBC-CBC linkage information (right) (FIG. 36G). Key statistics from this experiment follow:Total cells: 3,556Unique capsules: 362Multi-cell capsules (>5 cells): 204 (56.4%)Capsules with TBC linkage: 9858330968828SOMI-007 / 01WO - 352949-2034Transcriptional clusters: 7Cells per capsule: 1-62 (mean: 9.8, median: 6)Results

[0237] Analysis of 3,556 QC-filtered cells from 362 unique capsules revealed transcriptional heterogeneity across seven distinct clusters (FIG. 36A-B). Cluster identities included:Cluster 0 (n=1,518): GAPDH, TMSB10 - metabolically active cellsCluster 1 (n=681): MEIS2, CDH11 - mesodermal progenitorsCluster 2 (n=471): SOX2, SEMA6A - neural / pluripotent cellsCluster 3 (n=394): FAM184A, FOXA2 - endodermal lineageCluster 4 (n=219): ALPK2, HAS2 - matrix-producing cellsCluster 5 (n=152): RMRP, PRR9 - proliferating cellsCluster 6 (n=121): CER1, RHOBTB3 - anterior endoderm

[0238] Of 362 capsules, 204 (56.4%) contained 5 or more cells (FIG. 36E), demonstrating successful multi -cell capture per capsule. Cells from individual capsules co-localized in UMAP transcriptional space (FIG. 36A), confirming successful capsule-to-transcriptome association.

[0239] Furthermore, 98 multi-cell capsules demonstrated high-confidence TBC assignments (FIG. 36C), enabling trajectory-to-transcriptome linkage. Tire barcode rank plot (FIG. 36D) shows the characteristic "knee" inflection separating high-quality cells from background. Conclusions

[0240] These data demonstrate that the pool & split combinatorial barcoding method successfully associates single-cell transcriptomes with capsule identity. Tire co-localization of cells from the same capsule in transcriptional space, combined with successful TBC-CBC linkage, validates that: (1) cells within a single capsule share similar transcriptional programs reflecting their common microenvironment, (2) the multi-level barcode structure (CBC + TBC) enables both capsule identification and trajectory tracking at single-cell resolution, and (3) the method is compatible with diverse cell populations exhibiting distinct transcriptional states.Example 13. Data supporting single-cell transcriptome-to-capsule association

[0241] This example describes an experiment in which single-cell barcoding was performed by a droplet-based single cell profiling method according to single-cell track B (as shown in FIG. 27B), following trajectory barcoding and capsule barcoding.

[0242] Cells within SPCs that had undergone combinatorial barcoding rounds 1-2 (described in Example 8) were processed via Round 2.6 adapter ligation to append sequencing adapters to59330968828SOMI-007 / 01WG - 352949-2034both cellular cDNA and trajectory DNA. Round 2.6 adapter sequences used in this experiment are provided by sense sequences SEQ ID NOs: 197-212 and antisense sequence SEQ ID NO: 213, and are non-limiting examples. Following decapsulation with dextranase, cells were dissociated using TrypLE Express, filtered through a 40-micron strainer, and resuspended at high density (-33,000 cells / uL) in Ampligase reaction buffer. Cells were mixed with bridge oligonucleotides and thermostable ligase and encapsulated with beads with capture sequence barcodes into oil emulsion droplets. Thermocycling (12 cycles of 98°C for 30 seconds, 59°C for 2 minutes) ligated cellular barcodes at the beads to the pre-existing cDNA with appropriate barcode and overhang. Following emulsion breakage, template switching was performed with Maxima H Minus reverse transcriptase and TSO, then amplification was performed with a P5 primer and TSO primer. Libraries were prepared by enzymatic fragmentation followed by end repair and adapter ligation to affix a TruSeq Read2 adapter to the cDNA end. Then, PCR amplification was performed using a P5 primer and a P7-i7-Truseq Read2 primer to generate the sequencing library-. PCR amplification primers comprise a first sequence, an index sequence, and a second sequence. A non-limiting example of a P7 primer comprises a first sequence of SEQ ID NO: 216, an index sequence, and a second sequence of SEQ ID NO: 217. A non-limiting example of a P5 primer comprise a first sequence of SEQ ID NO: 218 or 220, an index sequence, and a second sequence of SEQ ID NO: 219 or 221. and P5 primers are provided by, respectively, SEQ ID NO: 216. Trajectory DNA was recovered separately from the decapsulation supernatant and amplified for the trajectory,' library’ using dedicated PCR primers targeting the PCR handle (RP1 sequence) at the Round 2.6 adapter as well as P5 Read 1 sequence.

[0243] Raw sequencing data was processed through a pipeline consisting of custom barcode error correction code, Kallisto (pseudoalignment), Bustools (UMI counting), and CellBender (ambient RNA removal), although any computational pipeline could be adapted to the librarystructure. The cellular barcode consists of capsule barcode (combinatorial sequences of CBC3, CBC2, CBCl) as well as the cellular barcode (CBC4) from the bead. Quality control filtering retained cells with >300 UMI, >100 detected genes, and <20% mitochondrial reads. UMAP visualization was performed with 20 principal components and 15 nearest neighbors. Trajectory Barcode (TBC) data from a dedicated TxC library,' was integrated via capsule ID matching.Data60330968828SOMI-007 / 01WG - 352949-2034

[0244] FIG. 37A provides UMAP visualization of single-cell transcriptomes from a droplet-based capsule-derived single cell experiment. Each panel highlights cells (black dots) originating from individual capsules containing 5 or more cells, with remaining cells shown in grey. Capsule Barcodes (CBC) are indicated as CBC3 CBC2 CBC1 well positions (e.g., " C9__F12JD3"). The co-localization of cells from the same capsule in UMAP space reflects shared transcriptional programs among cells captured within individual droplets, n = 824 total cells from 231 unique capsules; 49 capsules contained >5 cells.

[0245] FIG. 37B provides UMAP visualization of transcriptional cluster analysis, in which unsupervised clustering of single-cell transcriptomes reveals four distinct cell populations. “All Clusters” shows all seven clusters with differential grey shading. Each population is shown in a separate panel, with the individual cluster highlighting with top two marker genes identified by Wilcoxon rank-sum test.

[0246] FIG. 37C provides UMAP visualization demonstrating linkage between trajectory’ barcode (TBC) data and capsule barcode (CBC) data enabling linkage between treatment history (trajectory label) and single-cell legel gene expression profiles. The left panel provides an overview showing two representative capsules with confirmed TBC assignments, and the center and right panels show individual capsules displaying their TBC signatures ((TBC1 / TBC2 / TBC3 / TBC4 format from dedicated trajectory library) linked to their corresponding CBC identifiers (CBC3 / CBC2 / CBC1). The four-component TBC (TBC1 xx / TBC2.xx / TBC3 xx / TBC4 xx) provides high-complexity barcoding for unique capsule identification, while the CBC represents the physical well position encoding (CBC3 CBC2 CBC1).

[0247] FIGs. 37D provides a log -log visualization of cellular barcode quality assessment. X- axis shows barcode rank (ordered by decreasing UMI count), Y-axis shows total UMI count per barcode. The characteristic "knee" shape distinguishes true cells from empty droplets and ambient RNA. Dashed lines indicate the QC threshold (>300 UMI), yielding n=820 cells passing filter from 5,668 total barcodes with detectable UMI.

[0248] FIG. 37E provides a histogram showing the distribution of cells per capsule (CBC) after QC filtering. Total of 231 unique capsule IDs were identified, with cell counts ranging from 1 to 49 per capsule (mean: 3.6, median: 2). Vertical lines indicate mean (solid), median (dashed), and multi-cell threshold (dotted, >5 cells). 49 capsules (21%) contained >5 cells, qualifying as multi-cell capsules suitable for intra-capsule transcriptional analysis.

[0249] FIG. 37F provides a histogram showing the distribution of Capsule Barcode (CBC) associations per bead barcode across all CellBender-called cells (n=5,669, before UMI 61330968828filtering). Of 3,305 unique bead barcodes with valid CBC assignments, 2,922 (88.4%) were associated with a single CBC (capsule), while 383 (11.6%) were found in multiple capsules (2- 7 CBC combinations), suggesting mild overloading. The mild overloading suggests more capacity for cellular profiling with the droplet procedure by increasing the overloading factor. Mean CBC per bead barcode = 1.17.

[0250] FIGs. 37G-37H provide a consolidated visualization of droplet-based capsule-derived single-cell analysis workflow. Six representative multi-cell capsules showing CBC identifiers and cell counts demonstrate successful multi-cell capture per capsule (FIG. 37G). A cluster overview shows four transcriptionally distinct populations (left); a cluster legend with cell counts and top marker genes for biological interpretation; and two representative capsules with integrated TBC-CBC linkage demonstrating lineage tracking capability (right) (FIG. 37H). Key statistics from this experiment follow:Total cells: 824Unique capsules: 231Multi-cell capsules (>5 cells): 49 (21%)Capsules with TBC linkage: 23Transcriptional clusters: 4Cells per capsule: 1-49 (mean: 3.6, median: 2)BC4 specificity: 88.4% single-capsule assignmentResults

[0251] Analysis of 824 QC-filtered cells from 231 unique capsules revealed four distinct transcriptional clusters (FIG. 37A-B):Cluster 0 (n=590): LDHB, SET - 228 capsules representedCluster 1 (n=213): ACTB, CALR - 118 capsules representedCluster 2 (n=11): MALAT1, SFT2D2 - single-capsule outlier clusterCluster 3 (n=10): PEX13, ALDH18A1 - single-capsule outlier cluster

[0252] The barcode rank plot (FIG. 37D) demonstrates successful separation of true cells from background. Of 231 capsules, 49 (21%) contained 5 or more cells (FIG. 37E), reflecting the stochastic nature of droplet-based encapsulation. Cells from individual capsules co-localized in UMAP space, demonstrating successful transcriptome-to-capsule association (FIG. 37A). Twenty-three multi-cell capsules had confirmed TBC linkages (FIG. 37C). Analysis of cellular barcode specificity (FIG. 37F) showed modest level of overloading, which could be increased further given the pre-indexing of CBC sequences62330968828Conclusions

[0253] These data demonstrate that the droplet-based method successfully associates single¬ cell transcriptomes with capsule identity. The orthogonal barcoding system (CBC from pool-split rounds, CBC4 from beads) combined with trajectory barcode integration enables comprehensive tracking of cell identity, capsule membership, and treatment history (trajectory). The method is compatible with high-throughput single-cell processing while maintaining the trajectory tracking capability established during capsule culture.Example 14. Effects of adapter sequence GC% on capsule oligonucleotide hybridization and amplification of capsule label.

[0254] Capsule oligonucleotides comprising different 5’ adapter sequences were sequentially hybridized and ligated (top row) or amplified (bottom row) to produce a capsule label. FIG.38A shows the relative amount of the intended capsule label produced after each capsule oligonucleotide was added. The third capsule oligonucleotide (3BC, left) resulted in robust amplification of the capsule label (“major product”). The fourth capsule oligonucleotide (4BC, middle) resulted in dramatically lower amplification of the capsule label. Melting temperatures, GC%, and length of these adapter sequences are provided below in Table 1. The 5’ adapter of BC4 (BC3-BC4) has a lower GC% than the other capsule oligonucleotides, suggesting that GC content is important for adapter performance.Table 1.Adapter Melting GC% Length Temperature (°C)Stub-BCl 65.2 55 20BC1-BC2 64.5 50 20BC2-BC3 68.5 65 20BC3-BC4 62.6 50 20BC4-BC5 66.6 65 20

[0255] FIG. 38B shows sequencing data demonstrating relative efficiency of capsule label sequencing. Sequencing data support low efficiency of BC3-BC4 adapter. FIG. 38C provides data showing the relative amount of the intended capsule label produced after each casule oligonucleotide 'as added, comparing the previous BC3-BC4 adapter (“Old”) to a new' version 63330968828SOMI-007 / 01WG - 352949-2034with a 64.1 % GC content (“Mew”). The lower-GC adapter sequence yields a high amount of partial product (“3BC partial product”) relative to the amount of the desired product (“Desired Band’), while the higher-GC adapter sequence yields dramatically less partial product relative to the amount of desired product. These data indicate that higher GC% improves efficacy of adapters sequences in this method.Table 2. Illustrative trajectory oligonucleotides, comprising a combination of two sequences (combination number, listed in the left column, identifies a pair of sequences listed in columns “1” and “2”, identified by their SEQ ID NOs).SEQ ID NOs | SEQ ID NOs SEQ ID NOs | SEQ ID NOs | Comb. 1 2 Comb. 1 9 Comb. 1 2 Comb. 1 9 1 225 321 241 561 657 481 993 897 721 1329 1233 2 226.322 242 562 658 482 994 898 722 1330 1234 3 227 323 243 563 659 483 995 899 723 1331 1235 4 228 324 244 564 660 484 996 900 724 1332 1236 5 229 325 245 565 661 485 997 901 725 1333 1237 6 230 326 246 566 662 486 998 902 726 1334 1238 7 231 327 247 567 663 487 999 903 727 1335 1239 8 232 328 248 568 664 488 1000 904 728 1336 1240 9 2.33.329 249 569 665 489 1001 905 729 1337 1241 10 234 330 250 570 666 490 1002 906 730 1338 1242 11 235 331 251 571 667 491 1003 907 731 1339 1243 12 236 332 252 572 668 492 1004 908 732 1340 1244 13 237 333 253 573 669 493 1005 909 733 1341 1245 14 238 334 254 574 670 494 1006 910 734 1342 1246 15 239 335 255 575 671 495 1007 911 735 1343 1247 16 240.336 256 576 672 496 1008 912 736 1344 1248 17 241 337 257 577 673 497 1009 913 737 1345 1249 18 242 338 258 578 674 498 1010 914 738 1346 1250 19 243 339 259 579 675 499 1011 915 739 1347 1251 20 244 340 260 580 676 500 1012 916 740 1348 1252 21 245 341 261 581 677 501 1013 917 741 1349 1253 22 246 342 262 582 678 502 1014 918 742 1350 1254 23 247.343 263 58.3 679 503 1015 919 743 1351 1255 24 248 344 264 584 680 504 1016 920 744 1352 1256 25 249 345 265 585 681 505 1017 921 745 1353 1257 26 250 346 266 586 682 506 1018 922 746 1354 1258 27 251 347 267 587 683 507 1019 923 747 1355 1259 28 252 348 268 588 684 508 1020 924 748 1356 1260 29 253 349 269 589 685 509 1021 925 749 1357 1261 30 254 350 270 590 686 510 1022 926 750 1358 1262 31 255 351 271 591 687 511 1023 927 751 1359 1263 32 256 352 272 592 688 512 1024 928 752 1360 1264 33 257 353 273 593 689 513 1025 929 753 1361 1265 34 258 354 274 594 690 514 1026 930 754 1362 1266 35 259 355 275 595 691 515 1027 931 755 1363 1267 36 260 356 276 596 692 516 1028 932 756 1364 1268 37 261 357 277 597 693 517 1029 933 757 1365 1269 38 262 358 278 598 694 518 1030 934 758 1366 1270 39 263 359 279 599 695 519 1031 935 759 1367 127140 264 360 280 600 696 520 1032 936 760 1368 127264330968828SEQ ID NOs | SEQ ID NOs | SEQ ID NOs | SEQ ID NOs i Comb. 1 2 Comb. 1 2 Comb. 1 2 Comb. 1 2 41 265 361 281 601 697 521 1033 937 761 1369 1273 42 266 362 282 602 698 522 1034 938 762 1370 1274 43 267 363 283 603 699 523 1035 939 763 1371 1275 44 268 364 284 604 700 524 1036 940 764 1372 1276 45 269 365 285 605 701 525 1037 941 765 1373 1277 46 270 366 286 606 702 526 1038 942 766 1374 1278 47 271 367 287 607 703 527 1039 943 767 1375 1279 48 272 368 288 608 704 528 1040 944 768 1376 1280 49 273 369 289 705 609 529 1041 945 769 1377 1437 50 274 370 290 706 610 530 1042 946 770 1378 1438 51 275 371 291 707 611 531 1043 947 771 1379 1439 52 276 372 292 708 612 532 1044 948 772 1380 1440 53 277 373 293 709 613 533 1045 949 773 1381 1441 54 278 374 294 710 614 534 1046 950 774 1382 1442 55 279 375 295 711 615 535 1047 951 775 1383 1443 56 280 376 296 712 616 536 1048 952 776 1384 1444 57 281 377 297 713 617 537 1049 953 777 1385 1445 58 282 378 298 714 618 538 1050 954 778 1386 1446 59 283 379 299 715 619 539 1051 955 779 1387 1447 60 284 380 300 716 620 540 1052 956 780 1388 1448 61 285 381 301 717 621 541 1053 957 781 1389 1449 62 286 382 302 718 622 542 1054 958 782 1390 1450 63 287 383 303 719 623 543 1055 959 783 1391 1451 64 288 384 304 720 624 544 1056 960 784 1392 1452 65 289 385 305 721 625 545 1057 961 785 1393 1453 66 290 386 306 722 626 546 1058 962 786 1394 1454 67 291 387 307 723 627 547 1059 963 787 1395 1455 68 292 388 308 724 628 548 1060 964 788 1396 1456 69 293 389 309 725 629 549 1061 965 789 1397 1457 70 294 390 310 726 630 550 1062 966 790 1398 1458 71 295 391 311 727 631 551 1063 967 791 1399 1459 72 296 392 312 728 632 552 1064 968 792 1400 1460 73 297 393 313 729 633 553 1065 969 793 1401 1461 74 298 394 314 730 634 554 1066 970 794 1402 1462 75 299 395 315 731 635 555 1067 971 795 1403 1463 76 300 396 316 732 636 556 1068 972 796 1404 1464 77 301 397 317 733 637 557 1069 973 797 1405 1465 78 302 398 318 734 638 558 1070 974 798 1406 1466 79 303 399 319 735 639 559 1071 975 799 1407 1467 80 304 400 320 736 640 560 1072 976 800 1408 1468 81 305 401 321 737 641 561 1073 977 801 1409 1469 82 306 402 322 738 642 562 1074 978 802 1410 1470 83 307 403 323 739 643 563 1075 979 803 1411 1471 84 308 404 324 740 644 564 1076 980 804 1412 1472 85 309 405 325 741 645 565 1077 981 805 1413 1473 86 310 406 326 742 646 566 1078 982 806 1414 1474 87 311 407 327 743 647 567 1079 983 807 1415 1475 88 312 408 328 744 648 568 1080 984 808 1416 1476 89 313 409 329 745 649 569 1081 985 809 1417 1477 90 314 410 330 746 650 570 1082 986 810 1418 1478 91 315 411 331 747 651 571 1083 987 811 1419 1479 92 316 412 332 748 652 572 1084 988 812 1420 1480 93 317 413 333 749 653 573 1085 989 813 1421 1481 94 318 414 334 750 654 574 1086 990 814 1422 1482 95 319 415 335 751 655 575 1087 991 815 1423 148396 320 416 336 752 656 576 1088 992 816 1424 148465330968828SEQ ID NOs | SEQ ID NOs | SEQ ID NOs | SEQ ID NOs i Comb. 1 2 Comb. 1 2 Comb. 1 2 Comb. 1 2 97 417 321 337 753 657 577 1089 1185 817 1425 1485 98 418 322 338 754 658 578 1090 1186 818 1426 1486 99 419 323 339 755 659 579 1091 1187 819 1427 1487 100 420 324 340 756 660 580 1092 1188 820 1428 1488 101 421 325 341 757 661 581 1093 1189 821 1429 1489 102 422 326 342 758 662 582 1094 1190 822 1430 1490 103 423 327 343 759 663 583 1095 1191 823 1431 1491 104 424 328 344 760 664 584 1096 1192 824 1432 1492 105 425 329 345 761 665 585 1097 1193 825 1433 1493 106 426 330 346 762 666 586 1098 1194 826 1434 1494 107 427 331 347 763 667 587 1099 1195 827 1435 1495 108 428 332 348 764 668 588 1100 1196 828 1436 1496 109 429 333 349 765 669 589 1101 1197 829 1437 1497 110 430 334 350 766 670 590 1102 1198 830 1438 1498 111 431 335 351 767 671 591 1103 1199112 432 336 352 768 672 592 1104 1200113 433 337 353 769 673 593 1105 1201114 434 338 354 770 674 594 1106 1202115 435 339 355 771 675 595 1107 1203116 436 340 356 772 676 596 1108 1204117 437 341 357 773 677 597 1109 1205118 438 342 358 774 678 598 1110 1206119 439 343 359 775 679 599 1111 1207120 440 344 360 776 680 600 1112 1208121 441 345 361 777 681 601 1113 1209122 442 346 362 778 682 602 1114 1210123 443 347 363 779 683 603 1115 1211124 444 348 364 780 684 604 1116 1212125 445 349 365 781 685 605 1117 1213126 446 350 366 782 686 606 1118 1214127 447 351 367 783 687 607 1119 1215128 448 352 368 784 688 608 1120 1216129 449 353 369 785 689 609 1121 1217130 450 354 370 786 690 610 1122 1218131 451 355 371 787 691 611 1123 1219132 452 356 372 788 692 612 1124 1220133 453 357 373 789 693 613 1125 1221134 454 358 374 790 694 614 1126 1222135 455 359 375 791 695 615 1127 1223136 456 360 376 792 696 616 1128 1224137 457 361 377 793 697 617 1129 1225138 458 362 378 794 698 618 1130 1226139 459 363 379 795 699 619 1131 1227140 460 364 380 796 700 620 1132 1228141 461 365 381 797 701 621 1133 1229142 462 366 382 798 702 622 1134 1230143 463 367 383 799 703 623 1135 1231144 464 368 384 800 704 624 1136 1232145 465 369 385 801 897 625 1137 1233146 466 370 386 802 898 626 1138 1234147 467 371 387 803 899 627 1139 1235148 468 372 388 804 900 628 1140 1236149 469 373 389 805 901 629 1141 1237150 470 374 390 806 902 630 1142 1238151 471 375 391 807 903 631 1143 1239152 472 376 392 808 904 632 1144 124066330968828SEQ ID NOs | SEQ ID NOs | SEQ ID NOs | SEQ ID NOs i Comb. 1 2 Comb. 1 2 Comb. 1 2 Comb. 1 2 153 473 377 393 809 905 633 1145 1241154 474 378 394 810 906 634 1146 1242155 475 379 395 811 907 635 1147 1243156 476 380 396 812 908 636 1148 1244157 477 381 397 813 909 637 1149 1245158 478 382 398 814 910 638 1150 1246159 479 383 399 815 911 639 1151 1247160 480 384 400 816 912 640 1152 1248161 481 385 401 817 913 641 1153 1249162 482 386 402 818 914 642 1154 1250163 483 387 403 819 915 643 1155 1251164 484 388 404 820 916 644 1156 1252165 485 389 405 821 917 645 1157 1253166 486 390 406 822 918 646 1158 1254167 487 391 407 823 919 647 1159 1255168 488 392 408 824 920 648 1160 1256169 489 393 409 825 921 649 1161 1257170 490 394 410 826 922 650 1162 1258171 491 395 411 827 923 651 1163 1259172 492 396 412 828 924 652 1164 1260173 493 397 413 829 925 653 1165 1261174 494 398 414 830 926 654 1166 1262175 495 399 415 831 927 655 1167 1263176 496 400 416 832 928 656 1168 1264177 497 401 417 833 929 657 1169 1265178 498 402 418 834 930 658 1170 1266179 499 403 419 835 931 659 1171 1267180 500 404 420 836 932 660 1172 1268181 501 405 421 837 933 661 1173 1269182 502 406 422 838 934 662 1174 1270183 503 407 423 839 935 663 1175 1271184 504 408 424 840 936 664 1176 1272185 505 409 425 841 937 665 1177 1273186 506 410 426 842 938 666 1178 1274187 507 411 427 843 939 667 1179 1275188 508 412 428 844 940 668 1180 1276189 509 413 429 845 941 669 1181 1277190 510 414 430 846 942 670 1182 1278191 511 415 431 847 943 671 1183 1279192 512 416 432 848 944 672 1184 1280193 513 609 433 849 945 673 1281 1185194 514 610 434 850 946 674 1282 1186195 515 611 435 851 947 675 1283 1187196 516 612 436 852 948 676 1284 1188197 517 613 437 853 949 677 1285 1189198 518 614 438 854 950 678 1286 1190199 519 615 439 855 951 679 1287 1191200 520 616 440 856 952 680 1288 1192201 521 617 441 857 953 681 1289 1193202 522 618 442 858 954 682 1290 1194203 523 619 443 859 955 683 1291 1195204 524 620 444 860 956 684 1292 1196205 525 621 445 861 957 685 1293 1197206 526 622 446 862 958 686 1294 1198207 527 623 447 863 959 687 1295 1199208 528 624 448 864 960 688 1296 120067330968828SOMI-007 / 01WG - 352949-2034SEQ ID NOs | SEQ ID NOs | SEQ ID NOs | SEQ ID NOs i Comb. 1 2 Comb. 1 2 Comb. 1 2 Comb. 1 2 209 529 625 449 865 961 689 1297 1201210 530 626 450 866 962 690 1298 1202211 531 627 451 867 963 691 1299 1203212 532 628 452 868 964 692 1300 1204213 533 629 453 869 965 693 1301 1205214 534 630 454 870 966 694 1302 1206215 535 631 455 871 967 695 1303 1207216 536 632 456 872 968 696 1304 1208217 537 633 457 873 969 697 1305 1209218 538 634 458 874 970 698 1306 1210219 539 635 459 875 971 699 1307 1211220 540 636 460 876 972 700 1308 1212221 541 637 461 877 973 701 1309 1213222 542 638 462 878 974 702 1310 1214223 543 639 463 879 975 703 1311 1215224 544 640 464 880 976 704 1312 1216225 545 641 465 881 977 705 1313 1217226 546 642 466 882 978 706 1314 1218227 547 643 467 883 979 707 1315 1219228 548 644 468 884 980 708 1316 1220229 549 645 469 885 981 709 1317 1221230 550 646 470 886 982 710 1318 1222231 551 647 471 887 983 711 1319 1223232 552 648 472 888 984 712 1320 1224233 553 649 473 889 985 713 1321 1225234 554 650 474 890 986 714 1322 1226235 555 651 475 891 987 715 1323 1227236 556 652 476 892 988 716 1324 1228237 557 653 477 893 989 717 1325 1229238 558 654 478 894 990 718 1326 1230239 559 655 479 895 991 719 1327 1231240 560 656 480 896 992 720 1328 1232Table 3. Illustrative trajectory oligonucleotides for trajectory-only barcoding methods, comprising a combination of two sequences (combination number, listed in the left column, identifies a pair of sequences listed in columns “1” and "‘2”, identified by their SEQ ID NOs).SEQ ID NOs j SEQ ID NOs | SEQ ID NOs | SEQ ID NOs | Comb. 1 2 Comb. 1 2 Comb. 1 2 Comb. 1 2 831 1499 321 961 1629 643 1091 1759 965 1221 1889 1444 832 1500 322 962 1630 644 1092 1760 966 1222 1890 1445 833 1501 323 963 1631 645 1093 1761 967 1223 1891 1446 834 1502 324 964 1632 646 1094 1762 968 1224 1892 1447 835 1503 325 965 1633 647 1095 1763 969 1225 1893 1448 836 1504 326 966 1634 648 1096 1764 970 1226 1894 1449 837 1505 327 967 1635 649 1097 1765 971 1227 1895 1450 838 1506 328 968 1636 650 1098 1766 972 1228 1896 1451 839 1507 329 969 1637 651 1099 1767 973 1229 1897 1452 840 1508 330 970 1638 652 1100 1768 974 1230 1898 1453 841 1509 331 971 1639 653 1101 1769 975 1231 1899 1454 842 1510 332 972 1640 654 1102 1770 976 1232 1900 1455 843 1511 333 973 1641 655 1103 1771 977 1233 1901 1456844 1512 334 974 1642 656 1104 1772 978 1234 1902 145768330968828845 1513 335 975 1643 657 1105 1773 979 1235 1903 1458 846 1514 336 976 1644 658 1106 1774 980 1236 1904 1459 847 1515 337 977 1645 659 1107 1775 981 1237 1905 1460 848 1516 338 978 1646 660 1108 1776 982 1238 1906 1461 849 1517 339 979 1647 661 1109 1777 983 1239 1907 1462 850 1518 340 980 1648 662 1110 1778 984 1240 1908 1463 851 1519 341 981 1649 663 1111 1779 985 1241 1909 1464 852 1520 342 982 1650 664 1112 1780 986 1242 1910 1465 853 1521 343 983 1651 665 1113 1781 987 1243 1911 1466 854 1522 344 984 1652 666 1114 1782 988 1244 1912 1467 855 1523 345 985 1653 667 1115 1783 989 1245 1913 1468 856 1524 346 986 1654 668 1116 1784 990 1246 1914 1469 857 1525 347 987 1655 669 1117 1785 991 1247 1915 1470 858 1526 348 988 1656 670 1118 1786 992 1248 1916 1471 859 1527 349 989 1657 671 1119 1787 1185 1249 1917 1472 860 1528 350 990 1658 672 1120 1788 1186 1250 1918 1473 861 1529 351 991 1659 673 1121 1789 1187 1251 1919 1474 862 1530 352 992 1660 674 1122 1790 1188 1252 1920 1475 863 1531 353 993 1661 675 1123 1791 1189 1253 1921 1476 864 1532 354 994 1662 676 1124 1792 1190 1254 1922 1477 865 1533 355 995 1663 677 1125 1793 1191 1255 1923 1478 866 1534 356 996 1664 678 1126 1794 1192 1256 1924 1479 867 1535 357 997 1665 679 1127 1795 1193 1257 1925 1480 868 1536 358 998 1666 680 1128 1796 1194 1258 1926 1481 869 1537 359 999 1667 681 1129 1797 1195 1259 1927 1482 870 1538 360 1000 1668 682 1130 1798 1196 1260 1928 1483 871 1539 361 1001 1669 683 1131 1799 1197 1261 1929 1484 872 1540 362 1002 1670 684 1132 1800 1198 1262 1930 1485 873 1541 363 1003 1671 685 1133 1801 1199 1263 1931 1486 874 1542 364 1004 1672 686 1134 1802 1200 1264 1932 1487 875 1543 365 1005 1673 687 1135 1803 1201 1265 1933 1488 876 1544 366 1006 1674 688 1136 1804 1202 1266 1934 1489 877 1545 367 1007 1675 689 1137 1805 1203 1267 1935 1490 878 1546 368 1008 1676 690 1138 1806 1204 1268 1936 1491 879 1547 369 1009 1677 691 1139 1807 1205 1269 1937 1492 880 1548 370 1010 1678 692 1140 1808 1206 1270 1938 1493 881 1549 371 1011 1679 693 1141 1809 1207 1271 1939 1494 882 1550 372 1012 1680 694 1142 1810 1208 1272 1940 1495 883 1551 373 1013 1681 695 1143 1811 1209 1273 1941 1496 884 1552 374 1014 1682 696 1144 1812 1210 1274 1942 1497 885 1553 375 1015 1683 697 1145 1813 1211 1275 1943 1498 886 1554 376 1016 1684 698 1146 1814 1212887 1555 377 1017 1685 699 1147 1815 1213888 1556 378 1018 1686 700 1148 1816 1214889 1557 379 1019 1687 701 1149 1817 1215890 1558 380 1020 1688 702 1150 1818 1216891 1559 381 1021 1689 703 1151 1819 1217892 1560 382 1022 1690 704 1152 1820 1218893 1561 383 1023 1691 897 1153 1821 1219894 1562 384 1024 1692 898 1154 1822 1220895 1563 385 1025 1693 899 1155 1823 1221896 1564 386 1026 1694 900 1156 1824 1222897 1565 387 1027 1695 901 1157 1825 1223898 1566 388 1028 1696 902 1158 1826 1224899 1567 389 1029 1697 903 1159 1827 1225900 1568 390 1030 1698 904 1160 1828 1226901 1569 391 1031 1699 905 1161 1829 1227902 1570 392 1032 1700 906 1162 1830 122869330968828903 1571 393 1033 1701 907 1163 1831 1229 904 1572 394 1034 1702 908 1164 1832 1230 905 1573 395 1035 1703 909 1165 1833 1231 906 1574 396 1036 1704 910 1166 1834 1232 907 1575 397 1037 1705 911 1167 1835 1233 908 1576 398 1038 1706 912 1168 1836 1234 909 1577 399 1039 1707 913 1169 1837 1235 910 1578 400 1040 1708 914 1170 1838 1236 911 1579 401 1041 1709 915 1171 1839 1237 912 1580 402 1042 1710 916 1172 1840 1238 913 1581 403 1043 1711 917 1173 1841 1239 914 1582 404 1044 1712 918 1174 1842 1240 915 1583 405 1045 1713 919 1175 1843 1241 916 1584 406 1046 1714 920 1176 1844 1242 917 1585 407 1047 1715 921 1177 1845 1243 918 1586 408 1048 1716 922 1178 1846 1244 919 1587 409 1049 1717 923 1179 1847 1245 920 1588 410 1050 1718 924 1180 1848 1246 921 1589 411 1051 1719 925 1181 1849 1247 922 1590 412 1052 1720 926 1182 1850 1248 923 1591 413 1053 1721 927 1183 1851 1249 924 1592 414 1054 1722 928 1184 1852 1250 925 1593 415 1055 1723 929 1185 1853 1251 926 1594 416 1056 1724 930 1186 1854 1252 927 1595 609 1057 1725 931 1187 1855 1253 928 1596 610 1058 1726 932 1188 1856 1254 929 1597 611 1059 1727 933 1189 1857 1255 930 1598 612 1060 1728 934 1190 1858 1256 931 1599 613 1061 1729 935 1191 1859 1257 932 1600 614 1062 1730 936 1192 1860 1258 933 1601 615 1063 1731 937 1193 1861 1259 934 1602 616 1064 1732 938 1194 1862 1260 935 1603 617 1065 1733 939 1195 1863 1261 936 1604 618 1066 1734 940 1196 1864 1262 937 1605 619 1067 1735 941 1197 1865 1263 938 1606 620 1068 1736 942 1198 1866 1264 939 1607 621 1069 1737 943 1199 1867 1265 940 1608 622 1070 1738 944 1200 1868 1266 941 1609 623 1071 1739 945 1201 1869 1267 942 1610 624 1072 1740 946 1202 1870 1268 943 1611 625 1073 1741 947 1203 1871 1269 944 1612 626 1074 1742 948 1204 1872 1270 945 1613 627 1075 1743 949 1205 1873 1271 946 1614 628 1076 1744 950 1206 1874 1272 947 1615 629 1077 1745 951 1207 1875 1273 948 1616 630 1078 1746 952 1208 1876 1274 949 1617 631 1079 1747 953 1209 1877 1275 950 1618 632 1080 1748 954 1210 1878 1276 951 1619 633 1081 1749 955 1211 1879 1277 952 1620 634 1082 1750 956 1212 1880 1278 953 1621 635 1083 1751 957 1213 1881 1279 954 1622 636 1084 1752 958 1214 1882 1280 955 1623 637 1085 1753 959 1215 1883 1438 956 1624 638 1086 1754 960 1216 1884 1439 957 1625 639 1087 1755 961 1217 1885 1440 958 1626 640 1088 1756 962 1218 1886 1441 959 1627 641 1089 1757 963 1219 1887 1442960 1628 642 1090 1758 964 1220 1888 144370330968828Equivalents

[0256] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0257] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0258] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0259] Tlie present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. Tins includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0260] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0261] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0262] Other aspects are set forth within the following claims.71330968828

Claims

SOMI-007 / 01WO – 352949-2034CLAIMS1. A method of labeling a plurality of nucleic acid molecules within a semi -permeable capsule (SPC), comprising:a) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in a trajectory label on the surface of the SPC and a nucleic acid sequence in at least one of the nucleic acids in the plurality of nucleic acids w ithin the SPC; andb) combining the SPC with a second capsule oligonucleotide comprising a second capsule barcode, wlrerein the second capsule oligonucleotide hybridizes with the first capsule oligonucleotide to produce a capsule label;wherein the capsule label uniquely identifies the SPC; andwherein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label.

2. The method of claim 1, wherein the nucleic acid sequence in the trajectory label and the nucleic acid sequence in at least one of the nucleic acids is a polyadenylation sequence.

3. A method of labeling a plurality of nucleic acid molecules within a semi-permeable capsule (SPC), comprising:a) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising the first capsule barcode, wherein the first capsule oligonucleotide hybridizes with a trajectory label on the surface of the SPC and the second capsule oligonucleotide hybridizes with a poly-adenylation sequence on at least one of the nucleic acids in the plurality of nucleic acids within the SPC; andb) combining the SPC with a plurality of third capsule oligonucleotides comprising a second capsule barcode, wherein the third capsule oligonucleotide hybridizes with the first and the second capsule oligonucleotides to produce a capsule label;wherein the capsule label uniquely identifies the SPC; andwherein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label.

4. The method of any one of claims 1-3, comprising sequentially combining the SPC w ith 3, 4, 5, 6 or more capsule oligonucleotides each comprising a capsule barcode.72330968828SOMI-007 / 01WG - 352949-20345. The method of any one of claims 1-4, wherein tire method further comprises, prior to step a)sequentially combining the SPC with a first trajectory oligonucleotide and a second trajectory oligonucleotide, wherein the first and the second trajectory' oligonucleotides each comprise a trajectory’ barcode, wherein the second trajectory’ oligonucleotide hybridizes to the first trajectory oligonucleotide to produce the trajectory’ label;wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed.

6. The method of claim 5, wherein the SPC is combined with 3, 4, 5, 6 or more trajectory oligonucleotides each comprising a trajectory barcode.

7. The method of claim 5 or 6, further comprising combining the SPC with a trajectory’ terminator oligonucleotide.

8. Tire method of claim 7, wherein the trajectory' terminator oligonucleotide comprises a first capsule barcode or a universal barcode.

9. The method of any’ one of claims 1-8, wherein the first and second trajectory’ oligonucleotides are partially double -stranded DNA oligonucleotides comprising a 3’ single¬ stranded adapter sequence, a double-stranded trajectory barcode sequence, and a 5’ singlestranded adapter sequence.

10. Tire method of any one of claims 6-8, wherein the trajectory’ terminator oligonucleotide is a single-stranded DNA oligonucleotide.

11. A method of labeling a plurality of nucleic acid molecules within a semi -permeable capsule (SPC), comprising:a) combining the SPC with a first trajectory oligonucleotide and a second trajectory’ oligonucleotide, wherein the first and the second trajectory' oligonucleotides each comprise a trajectory' barcode, wherein the second trajectory' oligonucleotide hybridizes with the first trajectory oligonucleotide to produce a trajectory label, and wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed; and73330968828SOMI-007 / 01WG - 352949-2034b) sequentially combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising a second capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in die trajectory label and die second oligonucleotide hybridizes with die first capsule oligonucleotide to produce a capsule label that uniquely identifies the SPC;wherein the SPC comprises the trajectory' label; andwherein the SPC and the plurality of nucleic acid molecules each comprise the capsule label.

12. Tire method of claim 11, wherein the nucleic acid sequence in the trajectory' label and die nucleic acid sequence in at least one of the nucleic acids is a polyadenylation sequence.

13. A method of labeling a plurality of nucleic acid molecules within a semi -permeable capsule (SPC), comprising:a) combining the SPC with a first trajectory' oligonucleotide and a second trajectory oligonucleotide, wherein the first and the second trajectory' oligonucleotides each comprise a trajectory' barcode, wherein the second trajectory' oligonucleotide hybridizes with the first trajectory' oligonucleotide to produce a trajectory' label, and wherein the trajectory / label identifies a set of experimental conditions to which the SPC as exposed; andb) combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising the first capsule barcode, wherein the first capsule oligonucleotide hybridizes w ith the trajectory label on the surface of the SPC and the second capsule oligonucleotide hybridizes with a poly-adenylation sequence on at least one of the nucleic acids in the plurality of nucleic acids within the SPC; andc) combining the SPC w ith a plurality of third capsule oligonucleotides comprising a second capsule barcode, wherein the third capsule oligonucleotide hybridizes with tire first and the second capsule oligonucleotides to produce a capsule label;wherein the capsule label uniquely identifies the SPC; andw herein the SPC and the plurality of nucleic acid molecules each comprise the capsule label.

14. Tire method of any one of claims 11-13, wherein the SPC is combined with 3, 4, 5, 6 or more trajectory oligonucleotides each comprising a trajectory barcode, wherein each74330968828SOMI-007 / 01WG - 352949-2034trajectory oligonucleotide hybridizes with the previous trajectory oligonucleotide to extend the trajectory label.

15. Tire method of any one of claims 11-14, wherein the first and second trajectory oligonucleotides are partially double-stranded DNA oligonucleotides comprising a 3' singlestranded adapter sequence, a double-stranded trajectory barcode sequence, and a 5’ singlestranded adapter sequence.

16. The method of claim 15, wherein the 5’ single-stranded adapter sequence of the second trajectory' oligonucleotide hybridizes with tire 3’ single-stranded adapter sequence of the first trajectory oligonucleotide to extend the trajectory label.

17. The method of any one of claims 11-16, wherein step (a) further comprises combining the SPC with a trajectory terminator oligonucleotide.

18. I'he method of claim 17, wherein the trajectory terminator oligonucleotide comprises a first capsule barcode or a universal barcode.

19. lire method of claim 17 or 18, wherein the trajectory terminator oligonucleotide is a single-stranded DNA oligonucleotide.

20. The method of any one of claims 11-19, further comprising prior to step (b), combining the SPC with a polynucleotide kinase and a ligase to produce a ligated trajectory label.

21. Tire method of claim 20, wherein the method comprises no more than one ligation step.

22. The method of any one of claims 5-21, wherein each trajectory barcode corresponds to a unique experimental condition.

23. The method of any one of claims 1-22, wherein the method is performed using a plurality of SPCs, wherein each SPC within the plurality is exposed to a unique combination of experimental conditions.75330968828SOMI-007 / 01WG - 352949-203424. The method of any one of claims 1-22, wherein the method is performed using a plurality of SPCs divided into multiple sub-pluralities, and wherein each sub-plurality of SPCs is exposed to a unique combination of experimental conditions.

25. The method of any one of claims 1-24, wherein tire method comprises a reverse transcriptase step whereby the first capsule oligonucleotide is attached to at least one of the plurality of nucleic acids using reverse transcriptase primers that comprise tire first capsule barcode.

26. Tire method of any one of claims 1-25, wherein the second capsule oligonucleotide is attached to the first capsule oligonucleotide using hairpin ligation.

27. The method of any one of claims 1-25, wherein the second capsule oligonucleotide is atached to the first capsule oligonucleotide using bridge-linker ligation.

28. The method of any one of claims 1-25, wherein the second capsule oligonucleotide is attached to the first capsule oligonucleotide using a USER enzyme reaction.

29. The method of any one of claims 1-28, wherein the method further comprises release of the trajectory label from the SPC.

30. The method of any one of claims 1-29, wherein the first trajectory oligonucleotide and the second trajectory oligonucleotide each further comprise a 3’ adapter sequence and a 5" adapter sequence.

31. The method of claim 30, wherein the 3’ adapter sequence in the first trajectory oligonucleotide is complementary to the 5’ adapter sequence in the second trajectory’ oligonucleotide.

32. The method of any one of claims 30 or 31, wherein the SPC comprises an anchor oligonucleotide comprising a single-stranded sequence complementary to the 5’ adapter sequence of the first trajectory oligonucleotide.76330968828SOMI-007 / 01WO - 352949-203433. The method of claim 32, wherein the each of the 5’ and 3’ adapter sequences of the first and second trajectory oligonucleotides are at least 16 nucleotides in length.

34. Tlie method of claim 32, wherein the each of the the 5’ and 3’ adapter sequences of the first and second trajectory oligonucleotides are between about 16 nucleotides and about 20 nucleotides in length.

35. The method of claim 32, wherein the each of the the 5’ and 3’ adapter sequences of the first and second trajectory oligonucleotides are 16, 17, 18, 19, or 20 nucleotides in length.

36. Tlie method of any one of claims 32-35, wherein the the 5’ and 3’ adapter sequences of the first and second trajectory oligonucleotides each comprise at least 55%, at least 60%, at least 65%, at least 70%, or 75% GC content,37. The method of any one of claims 32-36, wherein the adapter sequences of tire first trajectory oligonucleotide and the second trajectory oligonucleotide each comprise about 75% GC content.

38. The method of any one of claims 1-37, wherein the first capsule oligonucleotide and the second capsule oligonucleotide each further comprise two adapter sequences.

39. The method of claim 38, wherein the adapter sequences of the first capsule oligonucleotide and the second capsule oligonucleotide are each at least 6 nucleotides in length.

40. Tire method of any one of claims 5-39, wherein the first trajectory barcode and second trajectory barcode are each between about 16 nucleotides and about 20 nucleotides in length.

41. The method of any one of claims 1-40, wherein the first capsule barcode and second capsule barcode are each between about 16 nucleotides and about 20 nucleotides in length.

42. Tire method of any one of claims 1-41, wherein the method is performed using a plurality of SPCs each comprising a plurality of nucleic acids.77330968828SOMI-007 / 01WG - 352949-203443. The method of claim 42, comprising one or more rounds of pooling and splitting the plurality of SPCs into multiple culture vessels.

44. Tlie method of any one of claims 1-43, wherein the plurality of nucleic acids are comprised within one or more cells and wherein the method further comprises contacting a plurality of nucleic acids within a single cell with a first cellular oligonucleotide comprising a first cellular barcode,wherein the cellular barcode uniquely identifies the single cell.

45. Tire method of claim 44, wherein the method comprises splitting the single cells into separate vessels prior to contacting tlie plurality of nucleic acids within the single cell with the first cellular oligonucleotide.

46. The method of claim 44, wherein the method comprises attaching the plurality of nucleic acids within the single cell to barcoded beads prior to contacting the plurality of nucleic acids within the single cell with the first cellular oligonucleotide.

47. A method of labeling a semi-permeable capsule (SPC) and a plurality of nucleic acid molecules within a plurality of single cells, comprising:a) sequentially combining the SPC with a first trajectory oligonucleotide and a second trajectory oligonucleotide, wherein the first and the second trajectory oligonucleotides each comprise a trajectory barcode, wherein tlie second trajectory oligonucleotide hybridizes to the first trajectory oligonucleotide to produce a trajectory label, and wherein the trajectory label identifies a set of experimental conditions to which the SPC was exposed; andb) sequentially combining the SPC with a first capsule oligonucleotide comprising a first capsule barcode and a second capsule oligonucleotide comprising a second capsule barcode, wherein the first capsule oligonucleotide hybridizes with a nucleic acid sequence in the trajectory label and the second oligonucleotide hybridizes with the first capsule oligonucleotide to produce a capsule label that uniquely identifies the SPC; andc) combining a plurality of nucleic acids from a single cell with a first cellular oligonucleotide comprising a first cellular barcode, wherein the cellular barcode uniquely identifies the single cell;wherein the SPC comprises the trajectory label; and78330968828SOMI-007 / 01WG - 352949-2034wherein the SPC and at least one of the plurality of nucleic acid molecules each comprise the capsule label; andwherein at least one of the plurality of nucleic acid molecules from the single cell comprises the cellular barcode.

48. A method of identifying or optimizing a cell differentiation protocol comprising:(a) exposing a first SPC comprising a first plurality of cells, each comprising a first plurality of nucleic acid molecules to a first set of sequential experimental conditions,(i) wherein the first SPC is labeled with a first trajectory label comprising one or more trajectory barcodes that identify each experimental condition in the first set of experimental conditions,(ii) wherein the first SPC and the first plurality of nucleic acid molecules comprise a first capsule label that identifies the first SPC; and(iii) wherein the first plurality of nucleic acids comprise a cellular barcode that identifies a single cell in the first plurality of cells;(b) exposing a second SPC comprising a second plurality of cells each comprising a second plurality of nucleic acid molecules to a second set of sequential experimental conditions,(i) wherein the second SPC is labeled with a second trajectory label comprising one or more trajectory barcodes that identify each experimental condition in the second set of experimental conditions;(ii) wherein the second SPC and the first plurality of nucleic acid molecules comprise a second capsule label that identifies the second SPC; and(iii) wherein the second plurality of nucleic acids comprise a cellular barcode that identifies a single cell in the second plurality of cells;(c) determining gene expression signatures of the first plurality and second plurality of cells; and(d) correlating each gene expression signature -with the set of experimental conditions defined by tire trajectory barcodes to identify or optimize the differentiation protocol.49, A population of semi-permeable capsules (SPCs) comprising a plurality of nucleic acid molecules, wherein each SPC is labeled with a corresponding capsule label to the nucleic acid molecules therein; wherein each capsule label comprises a first capsule barcode and a second capsule barcode, wherein the combination thereof uniquely identifies each SPC.79330968828SOMI-007 / 01WO - 352949-203450. A population of labeled nucleic acids, wherein each nucleic acid comprises:(i) a capsule label; and(ii) a sequence corresponding to a gene transcript;wherein the capsule label comprises a combination of barcodes that uniquely identify a semi-permeable capsule (SPC) from which the transcript originated.

51. A kit comprising:(i) one or more trajectory oligonucleotides; and(ii) one or more capsule oligonucleotides.

52. The kit of claim 51, further comprising one or more cellular barcodes.

53. A partially double stranded oligonucleotide comprising:a 5’ single stranded adapter sequence that is between about 16 and 25 nucleotides in length and comprises between 50% and 75% GC content;a double stranded barcode sequence; anda 3’ single strand adapter sequence that is between about 16 and 25 nucleotides in length and comprises between 50% and 75% GC content.

54. The oligonucleotide of claim 53, wherein the 5’ and 3’ adapter are the same length.

55. The oligonucleotide of claim 53, wherein the 5’ and 3’ adapter are different lengths.

56. Tire oligonucleotide of any one of claims 53-55, wherein the 5’ and / or 3’ adapter is 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

57. The oligonucleotide of any one of claims 53-56, wherein the 5’ and / or 3’ adapter comprise 50%, 55%, 60%, 65%, 70%, or 75% GC content.

58. Tire oligonucleotide of any one of claims 53-57, wherein the barcode sequence corresponds to an experimental condition.8033096882859. The oligonucleotide of any one of claims 53-58, wherein the oligonucleotide is a DNA oligonucleotide.

60. A library of partially double stranded oligonucleotides, wherein the oligonucleotides are selected from any one of claims 53-59, and wherein each oligonucleotide in the library' has a unique barcode sequence,61. A method of labeling a particle, comprising:hybridizing a first oligonucleotide of any one of claims 53-59 to a ssDNA oligonucleotide affixed to the surface of the particle, wherein the 5 ’ single stranded adapter sequence of the first oligonucleotide hybridizes to the ssDNA oligonucleotide;hybridizing a second oligonucleotide of any one of claims 53-59 to the first oligonucleotide, wherein the 3’ single stranded adapter sequence of the first oligonucleotide hybridizes with the 5’ single stranded adapter sequence of the second oligonucleotide;hybridizing a third oligonucleotide of any one of claims 43-49 to the second oligonucleotide, wherein the 3' single stranded adapter sequence of the second oligonucleotide hybridizes with the 5’ single stranded adapter sequence of the third oligonucleotide;phosphorylating available 5’ ends of the hybridized oligonucleotides; and ligating the hybridized oligonucleotides to form a double-stranded nucleic acid label.81330968828

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