Methods of identifying a cellular nascent RNA transcript

• US 9,284,604 B2
• Filed: 05/22/2013
• Issued: 03/15/2016
• Est. Priority Date: 11/22/2010
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Weissman, Jonathan, Churchman, Lee Stirling
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US13/900,356
• Publication Number:
  US 20140030706A1
• Time in Patent Office: 1,028 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation

Abstract

Methods and compositions for identifying a cellular nascent RNA transcript are provided.

Claims

1. A method of identifying a cellular nascent RNA transcript, comprising:

(a) arresting transcription in a cell;

(b) purifying a native cellular nascent RNA transcript; and

(c) sequencing said native cellular nascent RNA transcript thereby identifying the cellular nascent RNA transcript;

wherein said native cellular nascent RNA transcript is purified as part of an RNA polymerase complex, wherein said cellular nascent RNA transcript does not comprise a labeled nucleotide and wherein said cellular nascent RNA transcript is not crosslinked to an RNA polymerase complex.

2. The method of claim 1, wherein said sequencing is performed starting at a 3′

-end of said native cellular nascent RNA transcript.

3. The method of claim 1, wherein said sequencing comprises reverse transcribing said native cellular nascent RNA transcript into a DNA transcript.

4. The method of claim 1, wherein the native cellular nascent RNA transcript is sequenced by a deep sequencing method.

5. The method of claim 1, wherein the native cellular nascent RNA transcript is sequenced by a pyrosequencing method, a Sanger sequencing method, an SBS sequencing method, or a HANS sequencing method.

6. The method of claim 1, wherein the arresting comprises lowering the temperature of the cell to 10°

C. or below.

7. The method of claim 6, wherein the arresting comprises contacting the cell with liquid nitrogen thereby forming a flash-frozen cell.

8. The method of claim 1, wherein the purifying comprises contacting the RNA polymerase complex with a DNAse thereby removing the DNA template.

9. The method of claim 8, wherein said one or more RNA polymerase complex proteins is an RNA polymerase.

10. The method of claim 1, wherein the native cellular nascent RNA transcript is an unstable transcript.

11. The method of claim 1, wherein the native cellular nascent RNA transcript is a sense transcript.

12. The method of claim 1, wherein the native cellular nascent RNA transcript is an antisense transcript.

13. The method of claim 1, wherein the cell is a prokaryotic cell.

14. The method of claim 1, wherein the cell is a eukaryotic cell.

15. The method of claim 14, wherein the cell is a human cell.

16. The method of claim 14, wherein the cell is a cancer cell.

17. The method of claim 1, wherein the RNA polymerase complex is derived from a virus.

18. The method of claim 1, wherein the cell is subject to a stimulus prior to arresting.

19. The method of claim 18, wherein the stimulus is a small molecule.

20. The method of claim 18, wherein the stimulus is an inhibitory nucleic acid.

21. The method of claim 18, wherein the stimulus is a protein molecule.