METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS AND SINGLE-CELL TRANSCRIPTOME PROFILING USING TEMPLATE SWITCHING REACTION

Abstract

This application discloses methods for cDNA synthesis with improved reverse transcription, template switching and preamplification to increase both yield and average length of cDNA libraries generated from individual cells. The new methods include exchanging a single nucleoside residue for a locked nucleic acid (LNA) at the TSO 3′ end, using a methyl group donor, and/or a MgCl2 concentration higher than conventionally used. Single-cell transcriptome analyses incorporating these differences have full-length coverage, improved sensitivity and accuracy, have less bias and are more amendable to cost-effective automation. The invention also provides cDNA molecules comprising a locked nucleic acid at the 3′-end, compositions and cDNA libraries comprising these cDNA molecules, and methods for single-cell transcriptome profiling.

1 - 20 . (canceled) 
     
     
         21 . A cDNA library produced by a method of comprising the steps of:
 annealing a cDNA synthesis primer to said RNA molecule and synthesizing a first cDNA strand to form an RNA-cDNA intermediate; and   conducting a reverse transcriptase reaction by contacting said RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein the TSO comprises a locked nucleic acid (LNA) at its 3′-end, under conditions suitable for extension of the first DNA strand that is complementary to the RNA molecule, rendering it additionally complementary to the TSO.   
     
     
         22 - 30 . (canceled) 
     
     
         31 . The cDNA library of  claim 21 , wherein said reverse transcription reaction is conducted in the presence of a methyl group donor and a metal salt. 
     
     
         32 . The cDNA library of  claim 31 , wherein said methyl group donor is betaine. 
     
     
         33 . The cDNA library of  claim 31 , wherein said metal salt is magnesium salt. 
     
     
         34 . The cDNA library of  claim 33 , wherein said magnesium salt has a concentration of at least 7 mM, at least 8 mM, or at least 9 mM. 
     
     
         35 . The cDNA library of  claim 21 , wherein said template switching oligonucleotide comprises at least one or two ribonucleotide residues and said LNA residue. 
     
     
         36 . The cDNA library of  claim 35 , wherein said at least one or two ribonucleotide residues are riboguanine. 
     
     
         37 . The cDNA library of  claim 21 , wherein said locked nucleic acid residue is selected from the group consisting of locked guanine, locked adenine, locked uracil, locked thymine, locked cytosine, and locked 5-methylcytosine. 
     
     
         38 . The cDNA library of  claim 21 , wherein said locked nucleic acid residue is locked guanine. 
     
     
         39 . The cDNA library of  claim 21 , wherein said locked nucleic acid residue is at the 3′-most position. 
     
     
         40 . The cDNA library of  claim 21 , wherein said template switching oligonucleotide comprises at the 3′-end three nucleotide residues characterized by formula rGrG+N, wherein +N represents a locked nucleotide residue. 
     
     
         41 . The cDNA library of  claim 40 , wherein said template switching oligonucleotide comprises rGrG+G. 
     
     
         42 . The cDNA library of  claim 31 , wherein said methyl group donor is betaine, and said metal salt is MgCl 2  at a concentration of at least 9 mM. 
     
     
         43 . The cDNA library of  claim 21 , wherein the method further comprises amplifying said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide using an oligonucleotide primer. 
     
     
         44 . The cDNA library of  claim 21 , wherein said template switching oligonucleotide is selected from the oligonucleotides in Table S2. 
     
     
         45 . The cDNA library of  claim 21 , wherein the cDNA is synthesized on beads comprising an anchored oligo-dT primer. 
     
     
         46 . The cDNA library of  claim 45 , wherein said oligo-dT primer comprises a sequence of 5″-AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3″, wherein “N” is any nucleoside base, and “V” is selected from the group consisting of “A”, “C” and “G”. 
     
     
         47 . The cDNA library of  claim 42 , wherein the method further comprises PCR preamplification, tagmentation, and final PCR amplification. 
     
     
         48 . The cDNA library of  claim 47 , wherein the PCR preamplification is conducted without purifying the cDNA obtained from reverse transcription reaction. 
     
     
         49 . The cDNA library of  claim 21 , wherein said RNA is total RNA in a cell.