ANALYSIS OF NUCLEIC ACIDS ASSOCIATED WITH SINGLE CELLS USING NUCLEIC ACID BARCODES
Provided herein are methods and compositions for analyzing nucleic acids associated with single cells using nucleic acid barcodes. According to some embodiments, a method for producing one or more polynucleotides of interest comprises: obtaining a plurality of RNAs associated with one or more samples, wherein the samples are obtained from one or more subjects, each RNA is associated with a single sample, and the RNAs associated with each sample are present in a separate reaction volume; adding an adapter molecule to the RNAs associated with each sample, wherein the adapter molecule is generated using an enzymatic reaction and comprises a universal priming sequence, a barcode sequence, and a binding site; and incorporating the barcode sequence into one or more polynucleotides associated with each sample, thereby producing the one or more polynucleotides of interest.
1 . A composition comprising a plurality of beads, wherein each bead in the plurality is attached to a plurality of barcode adaptors, each barcode adaptor including a single-stranded DNA sequence with a barcode sequence, a universal priming sequence, a unique molecular identifier (UMI), and an RNA binding site.
2 . The composition of claim 1 , wherein the RNA binding site is a polyT tract.
3 . The composition of claim 1 , wherein the RNA binding site comprises a sequence complementary to at least one sequence region in one or more mRNAs.
4 . The composition of claim 1 , wherein the barcode adaptor is attached to the bead via the 5′ end of the barcode adaptor.
5 . The composition of claim 1 , wherein the barcode adaptor is attached to the bead via a thiol group.
6 . The composition of claim 1 , wherein each barcode adaptor attached to the same bead comprises the same barcode sequence.
7 . The composition of claim 1 , wherein each bead in the plurality of beads is attached to a plurality of barcode adaptors including a barcode sequence that is different from the barcode sequence included in the plurality of barcode adaptors attached to a different bead in the plurality of beads.
8 . A method of producing a polynucleotide of interest, the method comprising:
a. providing a bead including a plurality of barcode adaptors, each barcode adaptor including a single-stranded DNA sequence with a barcode sequence, a universal priming sequence, a UMI, and an RNA binding site;
b. generating a aqueous droplet containing (i) bead from step (a); (ii) a lysis reagent; (iii) a reverse transcriptase enzyme; and (iv) a single cell;
c. lysing the cell to release RNA molecules,
d. performing reverse transcription on the RNA molecules from the cells to generate cDNA molecules including the barcode sequence;
e. collecting the cDNA molecules including the barcode sequences; and
f. sequencing the cDNA molecules including the barcode sequences.
9 . The method of claim 8 , wherein the aqueous droplet is a water/oil emulsion.
10 . The method of claim 8 , wherein the RNA binding site is a polyT tract.
11 . The method of claim 8 , wherein the RNA binding site comprises a sequence complementary to at least one of the RNA molecules.
12 . The method of claim 8 , wherein the barcode adaptor is attached to the bead via the 5′ end of the barcode adaptor.
13 . The method of claim 8 , wherein the barcode adaptor is attached to the bead via avidin, streptavidin, biotin, gold, a thiol group, a carboxyl group, an epoxy group, a hydroxyl group or any combination thereof.
14 . The method of claim 8 , wherein the method further comprises: interrogating the cell for a phenotype prior to analyzing the nucleic acid, including contacting the cell with a nucleic acid marker, said nucleic acid marker including a nucleic acid linked to a binder, wherein the binder binds to a cell surface protein.
15 . A method of analyzing nucleic acids associated with single cells, the method comprising:
a. providing a bead including a plurality of barcode adaptors, each barcode adaptor including: a barcode sequence, a universal priming sequence, a UMI, and an RNA binding site;
b. generating a aqueous droplet containing (i) the bead from step (a); (ii) a lysis reagent; (iii) a reverse transcriptase enzyme; and (iv) a single cell;
c. lysing the cell to release RNA molecules,
d. performing reverse transcription on the RNA molecules from the cells to generate cDNA molecules including the barcode sequence;
e. collecting the cDNA molecules including the barcode sequences; and
f. sequencing the cDNA molecules including the barcode sequences, wherein at least 10,000 RNA molecules are analyzed.
16 . The method of claim 15 , wherein the aqueous droplet is a water/oil emulsion.
17 . The method of claim 15 , wherein the RNA binding site is a polyT tract.
18 . The method of claim 15 , wherein the RNA binding site comprises a sequence complementary to at least one of the RNA molecules.
19 . The method of claim 15 , wherein the barcode adaptor is attached to the bead via the 5′ end of the barcode adaptor.
20 . The method of claim 15 , wherein the barcode adaptor is attached to the bead via avidin, streptavidin, biotin, gold, a thiol group, a carboxyl group, an epoxy group, a hydroxyl group or any combination thereof.
21 . The method of claim 15 , wherein the method further comprises: interrogating the cell for a phenotype prior to analyzing the nucleic acid, including contacting the cell with a nucleic acid marker, said nucleic acid marker including a nucleic acid linked to a binder, wherein the binder binds to a cell surface protein.