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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)
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.
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.
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.
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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