Oligonucleotides associated with antibodies
Disclosed herein include systems, methods, compositions, and kits for determining protein expression and gene expression simultaneously and for sample indexing. In some embodiments, an oligonucleotide associated with a cellular component-binding reagent (e.g., an antibody) comprises one or more of a unique molecular label sequence, a primer adapter, antibody-specific barcode sequence, an alignment sequence, and/or a poly(A) sequence. In some embodiments, the oligonucleotide is associated with the cellular component-binding reagent via a linker (e.g., 5AmMC12).
1. A composition comprising: a plurality of cellular component-binding reagents each associated with a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, wherein the cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets, and wherein the cellular component-binding reagent specific oligonucleotide comprises an alignment sequence adjacent to a poly(dA) region, wherein the alignment sequence comprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof.
2. The composition of claim 1 , wherein the alignment sequence is 5′ to the poly(dA) region.
3. The composition of claim 1 , wherein the cellular component-binding reagent specific oligonucleotide comprises one or more of:
(a) a first molecular label sequence;
(b) a linker, wherein the cellular component-binding reagent specific oligonucleotide is associated with the cellular component-binding reagent through the linker; and
(c) the sequence of a first universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof.
4. The composition of claim 3 , wherein the first molecular label sequence is 2-20 nucleotides in length.
5. The composition of claim 3 , wherein the first molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides are identical.
6. The composition of claim 3 , wherein the first molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides are different.
7. The composition of claim 3 , wherein the first universal primer is 5-50 nucleotides in length.
8. The composition of claim 3 , wherein the first universal primer comprises an amplification primer, a sequencing primer, or a combination thereof.
9. The composition of claim 8 , wherein the sequencing primer comprises a P7 sequencing primer.
10. The composition of claim 3 , wherein the linker comprises a carbon chain.
11. The composition of claim 10 , wherein the carbon chain comprises 2-30 carbons.
12. The composition of claim 3 , wherein the linker comprises 5′ amino modifier C12 (5AmMC12), or a derivative thereof.
13. A method for measuring cellular component expression in cells, comprising:
contacting a plurality of cellular component-binding reagents with a plurality of cells comprising a plurality of cellular component targets, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of the plurality of cellular component targets, and wherein the cellular component-binding reagent specific oligonucleotide comprises an alignment sequence adjacent to a poly(dA) region;
extending barcodes hybridized to the cellular component-binding reagent specific oligonucleotides, or extending barcodes hybridized to products of the cellular component-binding reagent specific oligonucleotides, to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier sequence, or a complementary sequence thereof, and a first molecular label sequence, or a complementary sequence thereof; and
obtaining sequence information of the plurality of labeled nucleic acids, a complementary sequence thereof, or a portion thereof to determine the number of copies of at least one cellular component target of the plurality of cellular component targets in one or more of the plurality of cells.
14. The method of claim 13 , comprising prior to extending barcodes:
partitioning the plurality of cells associated with the plurality of cellular component-binding reagents to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the cellular component-binding reagents; and
in the partition comprising the single cell, contacting a barcoding particle with the cellular component-binding reagent specific oligonucleotides, wherein the barcoding particle comprises a plurality of barcodes each comprising a target binding region and a first molecular label sequence, and wherein two barcodes of the plurality of barcodes comprise different first molecular label sequences.
15. The method of claim 13 , wherein
(a) the alignment sequence comprises a guanine, a cytosine, a thymine, a uracil, or a combination thereof;
(b) the alignment sequence comprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof; and/or
(c) the alignment sequence is 5′ to the poly(dA) region.
16. The method of claim 13 , wherein the cellular component-binding reagent specific oligonucleotide comprises one or more of:
(a) a second molecular label sequence;
(b) a linker, wherein the cellular component-binding reagent specific oligonucleotide is associated with the cellular component-binding reagent through the linker; and
(c) the sequence of a first universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof.
17. The method of claim 16 , wherein the number of unique first molecular label sequences and/or the number of unique second molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the one or more of the plurality of cells.