June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Transcriptome profiling of single rod photoreceptors in the Rd1 mouse
Author Affiliations & Notes
  • David M Wu
    Retina Service/Department of Ophthalmology, Massachusetts Eye and Ear Infirmary/Harvard Medical School, Boston, MA
    Department of Genetics, Harvard Medical School/HHMI, Boston, MA
  • Rory Kirchner
    Department of Biostatistics, Harvard School of Public Health, Boston, MA
  • Magali Soumillon
    Broad Institute, Cambridge, MA
  • Tarej Mikkelsen
    Broad Institute, Cambridge, MA
  • Connie L Cepko
    Department of Genetics, Harvard Medical School/HHMI, Boston, MA
  • Footnotes
    Commercial Relationships David Wu, None; Rory Kirchner, None; Magali Soumillon, None; Tarej Mikkelsen, None; Connie Cepko, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5499. doi:
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      David M Wu, Rory Kirchner, Magali Soumillon, Tarej Mikkelsen, Connie L Cepko; Transcriptome profiling of single rod photoreceptors in the Rd1 mouse. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5499.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: There can be significant heterogeneity across a degenerating retina. Being able to resolve transcriptome changes at the single cell level may allow us to better understand photoreceptor death in degenerating Rd1 retinas. However, single cell RNA-seq is technically demanding and expensive. The very small amount of mRNA in photoreceptors adds further challenges. Here, we present results from two different methods for single-cell RNA-seq on rod photoreceptors from rd1 retinas.

Methods: Retinas from Rd1 mice were electroporated at P0 with a plasmid encoding dsRed driven by a rhodopsin promoter, which is specifically expressed by rods. Retinas were dissociated into a single cell suspension and dsRed-expressing rods were hand-picked via suction pipet or flow cytometry. Cells were lysed, mRNA reverse-transcribed, and cDNA prepared for next-generation sequencing. The cells were processed via two different pathways - a PCR-based amplification method or SCRB-Seq (Single Cell RNA Barcoding and Sequencing), which has been used in other systems to economically profile high numbers of single cells.

Results: Transcriptome profiling was performed on single rods or small groups of rods. With the PCR-based amplification method, abundant rod-enriched genes were detected at high levels and lower abundancy transcripts unrelated to phototransduction were also detected. Virtually no cone, RPE, or Muller glia enriched genes were detected. With SCRB-seq, cost per cell was two orders of magnitude lower, but genes detected were around ten-fold lower. Phototransduction genes were detected in SCRB-seq transcriptomes, whereas lower abundancy transcripts were less well represented than with PCR-based amplification.

Conclusions: We have performed single-cell RNA-seq on rod photoreceptors using two methods. The resulting transcriptomes show an abundancy of phototransduction-related transcripts, as expected in this cell type, without significant contamination from other retinal cells types. PCR-based amplification has greater resolution for lower abundancy transcripts, but at a significantly higher cost, limiting number of replicates. SCRB-seq allows analysis of many more cells at much lower cost, but with less representation of lower abundancy transcripts. A combination of complementary strategies and continued refinement of these techniques may enable more sophisticated analysis of retinal degeneration at the single cell level.


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