Cell-type-specific RNA-Seq using genetic ablation

[drosophiliac]

My lab is studying a cell population in Drosophila that is difficult to isolate, but genetically tractable. We created some flies in which we have genetically ablated the cells of interest using the GAL4/UAS system to induce apoptosis. We have collected control and experimental samples in triplicate and performed a paired-end RNA-Seq experiment using the Illumina HiSeq platform.

We are now analyzing the data. My question is whether anyone has seen examples of this kind of approach before.

Since the cells in question make up only a small proportion of the isolated tissue, we expect that most of the down-regulated genes will be fairly specific to this particular cell type. Up-regulated genes will likely be related to the induction of apoptosis itself and the process of cleaning up the mess.

Has anyone tried this sort of approach on cell types whose expression profiles are otherwise difficult to assess.

I have tried searching Google Scholar for as many search terms as I can think of to identify papers doing something like this, but I have so far come up empty. I guess this either means I am a pioneer or an idiot.

Any comments or references would be helpful. Also warnings on things we will need to be aware of during data analysis.

[SNPsaurus]

This doesn't directly address your question about prior studies using genetic ablation of small populations of cells, but TU-tagging in Drosophila was meant to help identify changed transcripts in small sub-populations:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783170/

It may reference some studies more similar to what you are doing. Some studies like this were done using microarrays, like these that I'm an author on: http://www.sciencedirect.com/science...96627303002897 or http://www.sciencemag.org/content/297/5590/2270.full which ablated larger tissues (eye or germline).

I would be amazed if more of these weren't done with RNA-Seq, but sorry I can't think of any right now... seems like everyone is working on better single-cell transcriptomics.

[drosophiliac]

I was finally able to find this paper (http://dx.doi.org/10.1093/nar/gks671), which uses a genetically-encoded nuclear tag followed by FACS to isolate different populations of fly neurons for RNA-Seq. That group seems to have modified a technique originally used in Arabidopsis (described here: http://dx.doi.org/10.1016/j.devcel.2010.05.013). Still it is all cell sorting rather than the "whole tissue minus the cells of interest" approach we are using.

I think the reason people may not have done things like what we are trying before is the worry that the induction of apoptosis will generate too many artifacts. But our ablative method has been super quick and can maybe be pitched as more of an exploratory technique rather than a way of definitively determining the transcriptome of a specific cell type.

One additional point is that our cells are embedded in a lot of waxy cuticle, which is difficult to homogenize gently. I don't know for sure, but I think this would make the FACS sorting technique difficult because the homogenization step requires maintaining intact nuclei.