RNA interactome analysis
View Patent ↗A method of sample analysis is provided. In certain cases, the method comprises: a) cross-linking the contents of a cell using a heat stable crosslinking agent to produce cross-linked ribonucleotide complexes; b) fragmenting the cross-linked ribonucleotide complexes to produce complexes comprising protein, RNA fragments and, optionally, genomic DNA fragments; c) contacting the complexes with a plurality of non-overlapping oligonucleotides comprise an affinity tag and that are complementary to a specific target RNA of the cell under high stringency conditions that include high temperature; d) isolating complexes that contain the oligonucleotides using the affinity tag to produce isolated complexes; e) enzymatically releasing the protein, RNA fragments and/or the genomic DNA fragments from the isolated complexes to produce a released component, without reversing the crosslinking; and f) analyzing the released component.
1. A method of sample analysis comprising:
a) cross-linking the contents of a cell using a crosslinking agent to produce cross-linked ribonucleotide complexes;
b) fragmenting said cross-linked ribonucleotide complexes to produce complexes comprising protein, RNA fragments and genomic DNA fragments;
c) contacting said complexes with a plurality of non-overlapping oligonucleotides that comprise an affinity tag and that are complementary to a specific target RNA of said cell under high stringency conditions;
d) isolating complexes that contain said oligonucleotides using the affinity tag of said oligonucleotides to produce isolated complexes;
e) treating the isolated complexes with RNAse and a protease, thereby enzymatically releasing said genomic DNA fragments from said isolated complexes to produce released genomic DNA;
f) sequencing said released genomic DNA to identify an occupancy site for an RNA.
2. The method of claim 1 , wherein said crosslinking agent is glutaraldehyde or formaldehyde.
3. The method of claim 1 , wherein said fragmenting is done enzymatically, chemically or physically.
4. The method of claim 1 , wherein said fragmenting is done by sonicating.
5. The method of claim 1 , wherein said fragmenting produces fragments having an average size in the range of 100 bp to 500 bp in length.
6. The method of claim 1 , wherein said oligonucleotides hybridize to sites in said specific RNA that are spaced along said RNA by at least 2 nucleotides.
7. The method of claim 1 , wherein said oligonucleotides are in the range of 15 to 30 nucleotides in length and temperature matched.
8. The method of claim 1 , wherein said oligonucleotides hybridize to mRNA.
9. The method of claim 1 , wherein said oligonucleotides hybridize to non-coding RNA.
10. The method of claim 1 , wherein said oligonucleotides hybridize to lncRNA.
11. The method of claim 1 , wherein said high stringency conditions include an incubation at least 37° C. for at least 4 hours.
12. The method of claim 1 , wherein said oligonucleotides are biotinylated.
13. The method of claim 1 , wherein said isolating is done using a support that comprises a ligand for said affinity tag.
14. The method of claim 13 , wherein said support is streptavidin.