TOTAL RNA PROFILING OF BIOLOGICAL SAMPLES AND SINGLE CELLS
Methods and materials for preparing DNA complementary to poly(A)-minus RNA are provided. The method includes conducting a template switching reaction by contacting a RNA-cDNA intermediate with a template switching oligonucleotide (TSO), extending the cDNA strand to include sequence complementary to the TSO and degrading the TSO. The method is capable of assaying a broad spectrum of coding and non-coding RNA from a single cell.
1 . A method for preparing DNA complementary to poly(A)-minus RNA comprising the steps of:
i) treating the poly(A)-minus RNA with
(a) poly(A) polymerase (PAP) to add a 3′ poly(A) tail to the poly(A)-minus RNA, thereby producing poly(A)-plus RNA, or
(b) polynucleotide transferase to add a homopolynucleotide poly(N), where poly(N) is selected from poly(A), poly(C), poly(G) and poly(U), thereby producing poly(N)-plus RNA;
ii) annealing a cDNA synthesis primer comprising oligo(dT) to the poly(A)-plus RNA produced in (i)(a), or annealing a cDNA synthesis primer comprising oligo(dN′) to the poly(N)-plus RNA produced in (i)(b), wherein N′ [“N prime”] is a nucleotide that basepairs with N, and synthesizing a first cDNA strand, thereby producing an RNA-cDNA intermediate;
iii) conducting a template switching reaction by contacting the RNA-cDNA intermediate with a template switching oligonucleotide (TSO) under conditions suitable for extension of the first cDNA strand, rendering the first cDNA strand additionally complementary to the TSO, wherein the TSO comprises at least two (2) deoxyuridine nucleotides
iv) degrading the TSO.
2 . The method of claim 1 , further comprising (v) amplifying sequence from the first cDNA strand to produce a pool of amplicons.
3 . The method of claim 2 , further comprising (vi) depleting high-abundance RNA species from the pool of amplicons.
4 . The method of claim 3 , wherein the high-abundance RNA species comprises ribosomal RNA.
5 . The method of claim 1 , wherein in step (iv) the TSO is degraded by treatment with uracil DNA glycosylase (UDG), and optionally with a combination of UDG and endonuclease VIII.
6 . The method of claim 1 wherein in step (i)(a) a mixture comprising poly(A)-minus RNA and polyadenylated RNA is treated with poly(A) polymerase (PAP) to add a 3′ poly(A) tail to the poly(A)-minus RNA and to the polyadenylated RNA, thereby producing the poly(A)-plus RNA.
7 . The method of claim 6 , wherein the mixture comprises RNA from one single cell, optionally a human cell.
8 . The method of claim 7 , wherein the mixture comprises total RNA from a single cell.
9 . The method of claim 6 , wherein the mixture comprises RNA from human cells.
10 . The method of claim 2 , wherein the step of amplifying comprises associating the sequence from the first cDNA strand with one or more sequence elements selected from adaptors, indexing sequences, oligonucleotide binding sequences and barcodes.
11 . The method of claim 2 , further comprising sequencing amplicons produced in step (v).
12 . The method of claim 2 , wherein steps (i)-(iv) or steps (i)-(v) are carried out in the same compartment.
13 . (canceled)
14 . A template switching oligonucleotide (TSO) comprising at least two (2) deoxyuridine nucleotides, at least two ribonucleotide residues.
15 . The TSO of claim 14 that is 30 to 50 nucleotides in length and comprises three (3) to ten (10) deoxyuridine nucleotides.
16 . The TSO of claim 15 that comprises rGrG+G at its 3′ end, where +G is a locked nucleic acid.
17 . The TSO of claim 14 , wherein
i) the TSO has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 , or at least 10 deoxyuridine nucleotides;
ii) at least 10%, at least 15% or at least 20% of the nucleotides in the TSO are deoxyuridine; or
iii) deoxyuridine nucleotides are spaced or positioned in the TSO such that following fragmentation of the TSO by deamination of all uridine nucleotides no remaining fragment of the TSO is longer than 10 nucleotides, no remaining fragment of the TSO is longer than 9 nucleotides, or no remaining fragment of the TSO is longer than 8 nucleotides.
18 . A method of identifying or distinguishing human cell types based on the abundance of non-coding transcripts comprising (i) determining an expression profile of at least one, preferably at least two, poly(A)-minus RNA type selected from miscRNA, lncRNA, snoRNA, miRNA, snRNA and tRNA in a cell(s) and (ii) matching the profile to a reference profile characteristic of a human cell type.
19 . The method of claim 12 , wherein cell lysis prior to step (i) is carried out in the same compartment.
20 . The TSO of claim 16 , wherein the TSO is biotinylated at the 5′ terminus.