Published: September 26th, 2018
Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).
Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.
The Drosophila visual system is an outstanding model for studying the genetic basis of development and behavior. It comprises a stereotyped cellular architecture1 and an advanced genetic toolkit for manipulating specific cell types2,3. A major strength of this system is the ability to autonomously interrogate gene function in cell types of interest with single cell resolution, using genetic mosaic methods4,5. We sought to combine these genetic tools with recent advances in next generation sequencing to perform cell type-specific....
1. Planning Before the Experiment
A general scheme of the protocol is shown in Figure 1. The protocol is divided into three major parts: fly work, sample preparation, sequencing and data analysis. The "Planning before the experiment" session of the protocol is not included in the general scheme for simplicity.
A calendar of the major parts of the protocol.......
This protocol is simple and not technically difficult to execute, but there are several key steps that if overlooked will cause a considerable reduction in cellular yield. (Step 2.3.2.) It is crucial that crosses are healthy, and that the food does not dry out. Regular watering of crosses is essential to maximize the number of flies available for dissection that are of the right genotype and at the correct stage of development. How often crosses need to be watered will vary depending on the food used and the housing cond.......
This research was funded by the NINDS of the National Institutes of Health under award number K01NS094545, andgrants from the Lefler Center for the Study of Neurodegenerative Disorders. We acknowledge Liming Tan and Jason McEwan for valuable conversations.....
|Proteolytic enzyme blend
|Heat Inactivated Bovine Serum
|Schneider's Culture Medium
|RNeasy Micro Kit
|RNA purification kit
|SuperScript II Reverse Transcriptase
|Q5 High-Fidelity 2x Master Mix
|New England Biolabs
|MinElute PCR Purification Kit
|Nextera XT DNA Library Prepration Kit
|Nextera XT Index Kit
|Test Tube with Cell Strainer Snap Cap
|Bottle-Top Vacuum Filter Systems
|FACSAria Flow Cytometer
|HiSeq 2500 Sequencing System
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