HIGH THROUGHPUT TRANSCRIPTOME ANALYSIS

• US 20150307874A1
• Filed: 07/09/2015
• Published: 10/29/2015
• Est. Priority Date: 01/09/2013
• Status: Abandoned Application

• Current Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Original Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Inventors: Amit, Ido, JAITIN, Diego, Keren-Shaul, Hadas, Valadarsky, Liran
• Application Number:
  US14/795,039
• Publication Number:
  US 20150307874A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Kits and methods for single cell or multiple cell transcriptome analysis are provided. An adapter polynucleotide is disclosed which comprises a double-stranded DNA portion of 15 base pairs and no more than 100 base pairs with a 3′ single stranded overhang of at least 3 bases and no more than 10 bases, wherein said double stranded DNA portion is at the 5′ end of the polynucleotide and wherein the sequence of said 3′ single stranded overhang is selected from the group consisting of SEQ ID NOs: 1-8 and 9, wherein the 5′ end of the strand of said double-stranded DNA which is devoid of said 3′ single stranded overhang comprises a free phosphate.

Claims

1. A kit for transcriptome analysis comprising:

(i) a first oligonucleotide comprising a polydT sequence at its terminal 3′

end, a RNA polymerase promoter sequence at its terminal 5′

end and a barcode sequence positioned between said polydT sequence and said RNA polymerase promoter sequence, wherein said barcode sequence comprises a cell barcode and a molecular identifier;

(ii) a second oligonucleotide being a single stranded DNA having a free phosphate at its 5′

end;

(iii) a third oligonucleotide being a single stranded DNA which is fully complementary to said second oligonucleotide.

3. The kit of claim 1, further comprising a T4 RNA ligase and/or a reverse transcriptase.

4. The kit of claim 1, wherein said first, said second and said third oligonucleotide are each packaged in a separate container.

5. The kit of claim 1, wherein said second oligonucleotide has a blocking group at its 3′

end.

6. The kit of claim 1, wherein said second and said third oligonucleotide are between 10-50 nucleotides in length.

7. The kit of claim 1, wherein said second and said third oligonucleotide are between 15 and 25 nucleotides in length.

8. The kit of claim 1, wherein said first oligonucleotide is no longer than 100 nucleotides.

2. A method of preparing a cell for transcriptome sequencing comprising:

(a) incubating a plurality of RNA molecules with a reverse transcriptase enzyme and a first oligonucleotide comprising a polydT sequence at its terminal 3′

end, a RNA polymerase promoter sequence at its terminal 5′

end and a barcode sequence positioned between said polydT sequence and said RNA polymerase promoter sequence under conditions that allow synthesis of a single stranded DNA molecule from said RNA, wherein said barcode sequence comprises a cell barcode and a molecular identifier;

(b) synthesizing a complementary sequence to said single stranded DNA molecule so as to generate a double stranded DNA molecule;

(c) incubating said double stranded DNA molecule with a T7 RNA polymerase under conditions which allow synthesis of amplified RNA from said double stranded DNA molecule;

(d) fragmenting said amplified RNA into fragmented RNA molecules of about 200 nucleotides;

(e) incubating said fragmented RNA molecules with a ligase enzyme and a second oligonucleotide being a single stranded DNA and having a free phosphate at its 5′

end under conditions that allow ligation of said second oligonucleotide to said fragmented RNA molecules so as to generate extended RNA molecules; and

(f) incubating said extended RNA molecules with a third oligonucleotide being a single stranded DNA and which is complementary to said second oligonucleotide, thereby preparing the cell for transcriptome sequencing.

9. The method of claim 2, being performed on a plurality of cells within a tissue, wherein said barcode sequence indicates the identity of the cells of the tissue.

10. The method of claim 9, further comprising pooling said single stranded DNA molecules synthesized in step (a), said pooling being effected prior to step (b).

11. A kit for synthesizing cDNA from an RNA sample comprising a library of adapter polynucleotides, a polydT oligonucleotide which is coupled to a barcoding sequence and a reverse transcriptase comprising terminal Deoxynucleotidyl Transferase (TdT) activity, wherein each member of the library comprises a double-stranded DNA portion with a 3′

single stranded overhang, wherein said double-stranded DNA portion comprises 15 base pairs and no more than 100 base pairs, wherein said 3′

single stranded overhang comprises at least 3 bases and no more than 10 bases, wherein said double stranded DNA portion is at the 5′

end of the polynucleotide, wherein the 5′

end of the strand of said double-stranded DNA which is devoid of said 3′

single stranded overhang comprises a free phosphate, wherein the sequence of said double stranded portion of said each member of the library is identical, wherein said sequence of said 3′

single stranded overhang of each member of the library is non-identical.

12. The kit of claim 11, wherein said reverse transcriptase comprises Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT).

13. The kit of claim 11, further comprising at least one of the following components:

(i) a ligase;

(ii) a DNA polymerase;

(iii) MgCl₂ (iv) a PCR primer; and

(v) RNase H.

14. The kit of claim 13, wherein said polydT oligonucleotide is attached to a solid support.

15. The kit of claim 11, wherein the 5′

terminus of said polydT oligonucleotide comprises an RNA polymerase promoter sequence.

16. A method for generating cDNA, comprising the steps of:

(a) combining an RNA sample with a polydT oligonucleotide under conditions sufficient to allow annealing of said polydT oligonucleotide to mRNA in said RNA sample to produce a polydT-mRNA complex, wherein a 5′

end of said polydT oligonucleotide is coupled to a barcoding sequence;

(b) incubating said polydT-mRNA complex with a reverse transcriptase comprising terminal Deoxynucleotidyl Transferase (TdT) activity under conditions which permit template-dependent extension of said polydT to generate an mRNA-cDNA hybrid;

(c) contacting said mRNA-cDNA hybrid with Rnase H under conditions which allow generation of a single stranded cDNA molecule; and

(d) incubating said single stranded cDNA molecule with;

(i) an adapter polynucleotide comprising a double-stranded DNA portion with a 3′

single stranded overhang, wherein said double-stranded DNA portion comprises 15 base pairs and no more than 100 base pairs, wherein said 3′

single stranded overhang comprises at least 3 bases and no more than 10 bases, wherein said double stranded DNA portion is at the 5′

end of the polynucleotide, wherein the 5′

end of the strand of said double-stranded DNA which is devoid of said 3′

single stranded overhang comprises a free phosphate and wherein the sequence of said 3′

single stranded overhang is selected such that it is capable of hybridizing to the 3′

end of said single stranded DNA molecule; and

(ii) a ligase enzyme, under conditions which permit ligation of said adapter polynucleotide to said single stranded cDNA molecule, thereby generating the cDNA.

17. The method of claim 16, wherein said polydT oligonucleotide is attached to a solid support.

18. The method of claim 16, further comprising amplifying the quantity of RNA in said RNA sample prior to step (a).

19. The method of claim 18, wherein said amplifying is effected by:

(a) contacting said RNA with a polydT oligonucleotide having a RNA polymerase promoter sequence at its terminal 5′

end under conditions sufficient to allow annealing of said polydT oligonucleotide to said RNA to produce a polydT-mRNA complex;

(b) incubating said polydT-mRNA complex with a reverse transcriptase devoid of terminal Deoxynucleotidyl Transferase (TdT) activity under conditions which permit template-dependent extension of said polydT to generate an mRNA-cDNA hybrid;

(c) synthesizing a double stranded DNA molecule from said mRNA-cDNA hybrid; and

(d) transcribing RNA from said double stranded DNA molecule.

20. A method of amplifying a plurality of gene sequences in an RNA sample comprising:

(a) contacting the RNA sample with a polydT oligonucleotide and a reverse transcriptase under conditions that allow synthesis of single stranded DNA molecules from said RNA, wherein a 5′

end of said polydT oligonucleotide is coupled to a barcoding sequence which comprises a cell identifier and a unique molecular identifier, and wherein a 5′

end of said barcoding sequence is coupled to a predetermined DNA sequence; and

(b) performing a multiplex PCR reaction on said single stranded DNA molecules using primer pairs which amplify a plurality of sequences of interest, wherein a first primer of each of said primer pairs hybridizes to said single stranded DNA molecules at a position which corresponds to said predetermined DNA sequence and a second primer of each of said primer pairs hybridizes to said single stranded DNA molecules at a position which encodes a target gene of interest, wherein each of said second primers of said primer pairs are coupled to an identical DNA sequence, thereby amplifying the plurality of gene sequences in the RNA sample.

21. The method of claim 20, further comprising performing a PCR reaction using a single primer pair which further amplifies said plurality of sequences of interest, wherein a first primer of said single primer pair hybridizes to said predetermined DNA sequence and a second primer of said single primer pair hybridizes to said identical DNA sequence.

22. The method of claim 21, wherein said first primer of said single primer pair and said second primer of said single primer pair are coupled to sequencing adaptors.