DETERMINING 5' TRANSCRIPT SEQUENCES
Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. There are provided, in some embodiments, methods of 5′-based and 3′-based gene expression profiling. Immune repertoire profiling methods are also provided in some embodiments.
1 - 5 . (canceled)
6 . A kit comprising:
a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises a molecular label and a target-binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences; and
a template switching oligonucleotide comprising the target-binding region, or a portion thereof.
7 . The kit of claim 6 , comprising a DNA polymerase lacking at least one of 5′ to 3′ exonuclease activity and 3′ to 5′ exonuclease activity.
8 . The kit of claim 6 , comprising a reverse transcriptase.
9 . The kit of claim 8 , wherein the reverse transcriptase comprises a viral reverse transcriptase, wherein the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.
10 . The kit of claim 6 , wherein the template switch oligonucleotide comprises one or more 3′ ribonucleotides.
11 . The kit of claim 10 , wherein the 3′ ribonucleotides comprise guanine.
12 . The kit of claim 6 , comprising one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof.
13 . The kit of claim 7 , wherein the DNA polymerase comprises a Klenow Fragment.
14 . The kit of claim 6 , comprising a buffer, a cartridge, or both.
15 . The kit of claim 6 , comprising one or more reagents for a reverse transcription reaction and/or an amplification reaction.
16 . The kit of claim 6 , wherein the target-binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof.
17 . The kit of claim 6 , wherein the oligonucleotide barcode comprises an identical sample label and/or an identical cell label
18 . The kit of claim 17 , wherein each sample label and/or cell label of the plurality of oligonucleotide barcodes comprise at least 6 nucleotides.
19 . The kit of claim 6 , wherein each molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides.
20 . The kit of claim 6 , wherein the target-binding region of the oligonucleotide barcode comprises an oligo(dT) sequence at least 10 nucleotides in length, and wherein the template switching oligonucleotide comprises an oligo(dT) sequence at least 5 nucleotides in length.
21 . The kit of claim 6 , wherein at least one of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on, or enclosed or partially enclosed in, a synthetic particle.
22 . The kit of claim 21 , wherein the synthetic particle is a bead.
23 . The kit of claim 22 , wherein the bead comprises a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A/G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof.
24 . The kit of claim 21 , wherein the synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.
25 . The kit of claim 21 ,
wherein each of the plurality of oligonucleotide barcodes comprises a linker functional group,
wherein the synthetic particle comprises a solid support functional group, and
wherein the support functional group and the linker functional group are associated with each other, and wherein the linker functional group and the support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.