Abstract
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.