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 structure comprising: a plurality of single-stranded DNA barcode adaptors attached to a solid support, wherein each barcode adaptor in the plurality includes a barcode sequence, a universal priming sequence, and an RNA binding site.
2 . The structure of claim 1 , wherein the RNA binding site is a polyT tract.
3 . The structure 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 structure of claim 1 , wherein the barcode adaptor is attached to the solid support via the 5′ end of the barcode adaptor.
5 . The structure of claim 1 , wherein the barcode adaptor is attached to the solid support via a thiol group.
6 . A method for producing one or more polynucleotides of interest, the method comprising:
a. providing 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. obtaining a plurality of RNA molecules associated with one or more samples;
c. adding the plurality of adapter molecules to the RNA molecules associated with the sample,
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.
7 . The method of claim 6 , wherein the RNA molecules associated with a sample are in a separate reaction volume.
8 . The method of claim 6 , wherein the RNA binding site is a polyT tract.
9 . The method of claim 6 , wherein the RNA binding site comprises a sequence complementary to at least one of the RNA molecules.
10 . The method of claim 6 , wherein the barcode adaptor is attached to a solid support.
11 . The method of claim 10 , wherein the barcode adaptor is attached to the solid support via the 5′ end of the barcode adaptor.
12 . The method of claim 10 , wherein the barcode adaptor is attached to the solid support via avidin, streptavidin, biotin, gold, a thiol group, a carboxyl group, an epoxy group, a hydroxyl group or any combination thereof.