Enrichment and sequencing of RNA species
Provided herein is a method for making an cDNA library, comprising adding an affinity tag-labeled GMP to the 5′ end of targeted RNA species in a sample by optionally decapping followed by incubating the sample with an affinity tag-labeled GTP and a capping enzyme, enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag, reverse transcribing the enriched RNA to produce a population of cDNAs, and adding a tail to the 3′ end of the population of cDNAs using a terminal transferase, to produce an cDNA library.
1. A method for making a eukaryotic cDNA library, comprising:
(a) treating a sample comprising capped, eukaryotic RNA species with a decapping enzyme to produce a decapped eukaryotic RNA species;
(b) adding an affinity tag-labeled guanosine monophosphate (GMP) to the 5′ end of the decapped eukaryotic RNA species by incubating the decapped eukaryotic RNA species with an affinity tag-labeled guanosine triphosphate (GTP) and a capping enzyme;
(c) enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag;
(d) optionally adding a poly(A) tail in (a), (b), or (c);
(e) eluting the RNA that remains bound to the affinity matrix after (c) but prior to (f), wherein the eluting comprises eluting the affinity matrix with a buffer comprising a concentration of salt in the range of 150 mM-350 mM; and
(f) reverse transcribing the eluted RNA to produce a eukaryotic cDNA library.
2. The method of claim 1 , wherein the decapping enzyme of step (a) is a member of the histidine triad (HIT) superfamily of pyrophosphatases.
3. The method of claim 2 , wherein the decapping enzyme is scavenger decapping enzyme (DcpS), or an aprataxin (APTX).
4. The method of claim 3 , wherein the APTX decapping enzyme is Saccharomyces cerevisiae APTX or Schizosaccharomyces pombe APTX.
5. The method of claim 1 , wherein the decapping enzyme of step (a) is a member of the NUDIX family of hydrolytic enzymes.
6. The method of claim 5 , wherein the NUDIX decapping enzyme is NUDT12 or NUDT15.
7. The method according to claim 1 , wherein (c) further comprises selectively binding the RNA to streptavidin beads in a buffer containing 1 M-3 M salt.
8. The method according to claim 7 , wherein the salt in (c) is NaCl.
9. The method according to claim 1 , wherein the salt in (e) is NaCl.
10. The method according to claim 1 , wherein the method comprises, after (f) treating the produced eukaryotic cDNA library with a single strand specific RNAse.
11. The method of claim 1 , wherein (f) further comprises: adding a tail to the 3′ end of the produced eukaryotic cDNAs using a terminal transferase.
12. The method according to claim 11 , wherein the terminal transferase adds a tail comprising a plurality of Gs at the 3′ end of the cDNA.
13. The method according to claim 12 , wherein the plurality of Gs is in the range of 10 to 20 nucleotides.
14. The method according to claim 11 , wherein step (f) further comprises using a primer that hybridizes to the 3′ tails of the produced eukaryotic cDNAs to form a second strand cDNA library.
15. The method according to claim 1 , wherein step (f) further comprises using an oligo-dT primer.
16. The method according to claim 1 , wherein step (f) further comprises amplifying the produced cDNA library.
17. The method according to claim 16 , further comprising amplifying the cDNA library using primers that comprise deoxyuridine.
18. The method according to claim 17 , further comprising cleaving the product of amplification using a mix comprising uracil-N-deglycosylase (UNG) or uracil DNA glycosylase (UDG) to produce overhangs at the ends of the amplification products.
19. The method according to claim 16 , further comprising ligating a loop adapter to one or both ends of the cDNA.
20. A method of classifying the 5′ cap structure of a sample of capped eukaryotic RNA species, comprising:
(a) treating separate portions of the sample with different decapping enzymes and optionally treating one portion with no decapping enzyme;
(b) adding an affinity tag-labeled guanosine monophosphate to the 5′ end of the treated RNA species by incubating each portion with the affinity tag-labeled guanosine triphosphate (GTP) and a capping enzyme or a chemical capping agent;
(c) enriching for affinity tagged RNA species in each portion using an affinity matrix that binds to the affinity tag;
(d) separately reverse transcribing enriched RNA to produce corresponding cDNA libraries;
(e) sequencing the cDNA libraries; and
(f) comparing the sequences obtained from the cDNA libraries.