Compositions and methods for constructing cDNA libraries that allow for mapping the 5′ and 3′ ends of RNAs

• US 9,828,600 B2
• Filed: 09/22/2014
• Issued: 11/28/2017
• Est. Priority Date: 09/20/2013
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Moore, Melissa J., Heyer, Erin E., Ricci, Emiliano P., Ozadam, Hakan, Cenik, Can, Li, Xin
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/492,815
• Publication Number:
  US 20150099671A1
• Time in Patent Office: 1,163 Days
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

This disclosure provides methods and compositions for preparing and constructing cDNA libraries.

Claims

1. A method of optimizing the preparation of RNA molecules from a biological sample for sequencing, consisting essentially of the steps of:

providing a biological sample comprising RNA molecules;

ligating a first DNA adaptor to the 3′

end of the RNA molecules, wherein the ligating is performed under conditions that optimize the ligation reaction, wherein the conditions that optimize the ligation reaction comprise carrying out the reaction in the presence of about 470 nM of the first DNA adaptor and incubating the reaction at about 30°

C. for about 6 hours;

reverse transcribing the RNA molecules using a primer under conditions that optimize the reverse transcription reaction to produce single-stranded cDNA molecules, wherein the conditions that optimize the reverse transcription reaction comprise using about 5 units of SuperScript III reverse transcriptase and carrying out the reaction for about 30 mins at about 55°

C. in the absence of any additional MgCl₂, wherein the primer comprises a first portion that is complementary to the first DNA adaptor and a second portion that comprises a forward primer sequence joined to a reverse primer sequence by a flexible linker;

gel purifying the cDNA molecules;

circularizing the purified cDNA molecules under conditions that optimize the circularization reaction, wherein the conditions that optimize the circularization reaction comprise carrying out the reaction in the presence of all or essentially all of the cDNA molecules obtained after the purifying step in the reaction, in the presence of about 1M betaine, at about 60°

C. for about 3 hours;

amplifying the circularized cDNA molecules under conditions that optimize the amplification reaction, wherein the conditions that optimize the amplification reaction comprise carrying out the reaction in the presence of the circularization reaction at about 20% of the total reaction volume,

thereby preparing RNA molecules from a biological sample for sequencing.

2. A method of preparing mRNA molecules in a biological sample for sequencing, comprising:

providing capped mRNA molecules from the biological sample;

ligating a first DNA adaptor to the 3′

ends of the capped mRNA molecules;

ligating a unique RNA adaptor to the 5′

ends of de-capped mRNA molecules;

fragmenting the mRNA molecules and ligating a second DNA adaptor to the newly-formed 3′

ends of the fragmented mRNA molecules;

reverse transcribing the fragmented mRNA molecules to produce single-stranded complementary DNA (cDNA) molecules;

circularizing the single-stranded cDNA molecules; and

amplifying the circularized cDNA molecules,

thereby preparing the mRNA molecules in the biological sample for sequencing, wherein the sequencing captures both 5′ and

3′

ends of the mRNA molecules, and wherein the sequencing determines the length of the polyA tail of the mRNA molecules.

3. The method of claim 2, wherein the sequencing is deep sequencing.

4. The method of claim 2, wherein the sequencing is paired-end sequencing.

5. The method of claim 2, wherein the first DNA adaptor is pre-adenylated.

6. The method of claim 2, wherein the second DNA adaptor is pre-adenylated.

7. The method of claim 2, wherein the mRNA molecules are de-capped using tobacco acid pyrophosphatase (TAP).

8. The method of claim 2, wherein the mRNA molecules are fragmented using alkaline hydrolysis.

10. The method of claim 2, further comprising, after said ligating the unique RNA adaptor to the 5′

ends of the de-capped mRNA molecules, inactivating excess unique RNA adaptor not ligated to the 5′

ends of the de-capped mRNA molecules.

11. The method of claim 10, wherein said inactivating comprises oxidation of the excess unique RNA adaptor not ligated to the 5′

ends of the de-capped mRNA molecules.

12. The method of claim 5, further comprising, after said ligating the pre-adenylated first DNA adaptor to the 3′

ends of the capped mRNA molecules, inactivating excess pre-adenylated first DNA adaptor not ligated to the 3′

ends of the capped mRNA molecules.

13. The method of claim 12, wherein said inactivating comprises deadenylating the excess pre-adenylated first DNA adaptor not ligated to the 3′

ends of the capped mRNA molecules.

14. The method of claim 2, further comprising depleting non-coding RNAs from said biological sample.

15. The method of claim 14, wherein said depleting the biological sample of non-coding RNAs comprises contacting the biological sample with a mutant eIF4E cap-binding protein.

16. The method of claim 15, wherein said mutant eIF4E cap-binding protein comprises a GST tag.

9. A method of preparing non-coding RNA molecules in a biological sample for sequencing, comprising:

providing non-capped non-coding RNA molecules from the biological sample;

ligating a first DNA adaptor to the 3′

ends of the non-coding RNA molecules;

ligating a unique RNA adaptor to the 5′

ends of the non-coding RNA molecules;

fragmenting the non-coding RNA molecules and ligating a second DNA adaptor to the newly-formed 3′

ends of the fragmented non-coding RNA molecules;

reverse transcribing the fragmented non-coding RNA molecules to produce single-stranded complementary DNA (cDNA) molecules;

circularizing the single-stranded cDNA molecules; and

amplifying the circularized cDNA molecules, thereby preparing the non-coding RNA molecules in the biological sample for sequencing, wherein the sequencing captures both 5′ and

3′

ends of the non-coding RNA molecules.

17. The method of claim 9, further comprising, after said ligating the unique RNA adaptor to the 5′

ends of the non-coding RNA de capped RNA molecules, inactivating said unique RNA adaptor ligated to the 5′

ends of the non-coding RNA de-capped RNA molecules.

18. The method of claim 17, wherein said inactivating the unique RNA adaptor ligated to the 5′

ends of the non-coding RNA molecules comprises oxidation of the unique RNA adaptor ligated to the 5′

ends of the non-coding RNA molecules.

19. The method of claim 9, wherein the first DNA adaptor is pre-adenylated.

20. The method of claim 9, wherein the second DNA adaptor is pre-adenylated.

21. The method of claim 19, further comprising, after said ligating the pre-adenylated first DNA adaptor to the 3′

ends of the non-coding RNA capped RNA molecules, inactivating excess pre-adenylated first DNA adaptor not ligated to the 3′

ends of the non-coding RNA de-capped RNA molecules.

22. The method of claim 21, wherein said inactivating comprises deadenylating the excess pre-adenylated first DNA adaptor not ligated to the 3′

ends of the non-coding RNA molecules.