Visualizing SNVs to
quantify allele-specific
expression in single cells
Marshall J Levesque, Paul Ginart, Yichen Wei, Arjun...
Paternal RNA
Maternal RNA
Transcription
Sites
Nucleus
Cytoplasm
Heterozygotic cell
Maternal Paternal
Human cells produce R...
Human cells produce RNA
using two copies of a gene
Mutant Wild-type
Wild-type RNA
Mutant RNA
Transcription
Sites
Nucleus
C...
RNA FISH probes directly
detect RNA in single cells
RNA
FISH: Fluorescence in situ Hybridization
How do we distinguish
chromosomes and their RNA?
Chr. 19EEF2 mRNAEEF2 intron
Levesque and Raj, Nature Methods doi:10.1038/...
Our probes are not sensitive to
single nucleotide differences
RNA
Our probes are not sensitive to
single nucleotide differences
RNA
Our probes are not sensitive to
single nucleotide differences
RNA
A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C
Masked probes provide specificity
thru a sh...
A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C
C
T
A
G
G
T
C
T
G
T
T
G
G
A
T
C
C
A
G
A
C
...
C T T T A G C T A G G T C T G T T G
G A T C C A G A C A A C
A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U ...
A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C
C
T
A
G
G
T
C
T
G
T
T
G
G
A
T
C
C
A
G
A
C
...
A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C
C
T
A
G
G
T
C
T
G
T
T
G
G
A
T
C
C
A
G
A
C
...
C T T T G G C T A G G T C T G T T G
A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C
toehol...
RNA
Single oligos produce false positives
when bound to off-targets
RNA
Single oligos produce false positives
when bound to off-targets
Single oligos produce false positives
when bound to off-targets
Single oligo detection assay takes
advantage of co-localization
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
Single oligo detection assay takes
advantage of co-localization
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH clearly shows
genotype of melanoma cell lines
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic
imbalance in the cell population
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic
imbalance in single-cells
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic
imbalance in single-cells
p = 0.00017
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic
imbalance in single-cells
p = 0.083
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic
imbalance in single-cells
p = 0.83
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH + iceFISH distinguishes
maternal from paternal chromosome
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
...
SNP FISH + iceFISH distinguishes
maternal from paternal chromosome
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
Acknowledgements
Members of the Raj Lab
UPenn Bioengineering
Herlyn Lab
Wistar Institute
Biosearch Technologies
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Visualizing SNVs to quantify allele-specific expression in single cells

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We present a FISH-based method for detecting single- nucleotide variants (SNVs) in exons and introns on individual RNA transcripts with high efficiency. We used this method
to quantify allelic expression in cell populations and in single cells, and also to distinguish maternal from paternal chromosomes in single cells.

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Visualizing SNVs to quantify allele-specific expression in single cells

  1. 1. Visualizing SNVs to quantify allele-specific expression in single cells Marshall J Levesque, Paul Ginart, Yichen Wei, Arjun Raj Systems Biology Lab, UPenn Bioengineering Nature Methods (2013) doi:10.1038/nmeth.2589
  2. 2. Paternal RNA Maternal RNA Transcription Sites Nucleus Cytoplasm Heterozygotic cell Maternal Paternal Human cells produce RNA using two copies of a gene
  3. 3. Human cells produce RNA using two copies of a gene Mutant Wild-type Wild-type RNA Mutant RNA Transcription Sites Nucleus Cytoplasm Heterozygotic cell
  4. 4. RNA FISH probes directly detect RNA in single cells RNA FISH: Fluorescence in situ Hybridization
  5. 5. How do we distinguish chromosomes and their RNA? Chr. 19EEF2 mRNAEEF2 intron Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372
  6. 6. Our probes are not sensitive to single nucleotide differences RNA
  7. 7. Our probes are not sensitive to single nucleotide differences RNA
  8. 8. Our probes are not sensitive to single nucleotide differences RNA
  9. 9. A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C Masked probes provide specificity thru a short “toehold” sequence C T T T A G C T A G G T C T G T T G G A T C C A G A C A A C probe mask toehold dye RNA target
  10. 10. A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C C T A G G T C T G T T G G A T C C A G A C A A C C T T T A G Masked probes bind through DNA strand displacement probe m ask dye RNA target toehold
  11. 11. C T T T A G C T A G G T C T G T T G G A T C C A G A C A A C A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C Masked probes bind through DNA strand displacement probe mask dye RNA target toehold
  12. 12. A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C C T A G G T C T G T T G G A T C C A G A C A A C C T T T A G Competing masked probes distinguish single nucleotide variants probe m ask toehold RNA target dye
  13. 13. A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C C T A G G T C T G T T G G A T C C A G A C A A C C T T T G G RNA target probe m ask toehold C T T T A G C T A G G T C T G T T G G A T C C A G A C A A C dye Competing masked probes distinguish single nucleotide variants
  14. 14. C T T T G G C T A G G T C T G T T G A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C toehold RNA target G A T C C A G A C A A C mask probe dye C T T T A G C T A G G T C T G T T G G A T C C A G A C A A C Competing masked probes distinguish single nucleotide variants
  15. 15. RNA Single oligos produce false positives when bound to off-targets
  16. 16. RNA Single oligos produce false positives when bound to off-targets
  17. 17. Single oligos produce false positives when bound to off-targets
  18. 18. Single oligo detection assay takes advantage of co-localization Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  19. 19. Single oligo detection assay takes advantage of co-localization Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  20. 20. SNP FISH clearly shows genotype of melanoma cell lines Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  21. 21. SNP FISH shows mRNA allelic imbalance in the cell population Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  22. 22. SNP FISH shows mRNA allelic imbalance in single-cells Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  23. 23. SNP FISH shows mRNA allelic imbalance in single-cells p = 0.00017 Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  24. 24. SNP FISH shows mRNA allelic imbalance in single-cells p = 0.083 Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  25. 25. SNP FISH shows mRNA allelic imbalance in single-cells p = 0.83 Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  26. 26. SNP FISH + iceFISH distinguishes maternal from paternal chromosome Levesque et al, Nature Methods, doi:10.1038/nmeth.2589 Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372
  27. 27. SNP FISH + iceFISH distinguishes maternal from paternal chromosome Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
  28. 28. Acknowledgements Members of the Raj Lab UPenn Bioengineering Herlyn Lab Wistar Institute Biosearch Technologies

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