Purification of Low-abundant Cells in the Drosophila Visual System

Summary

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).

Abstract

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.

Introduction

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 types^2,3. 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 methods^4,5. We sought to combine these genetic tools with recent advances in next generation sequencing to perform cell type-specific....

Protocol

1. Planning Before the Experiment

1. Before starting the experiment, perform a small-scale pilot experiment to confirm if the genetics work as expected to specifically label the desired cells.
   1. As a first pass, use immunohistochemistry and light microscopy to determine if unwanted cells are labeled. Ideally, genetic markers will be specific within the retina and optic lobe (Figure 3). Only optic lobes are used for cell dissociation and so labeling in the central brain is not an issue. At the same time, determine how many cells of interest are labeled per brain.
   2. If the genetic label(s) have t....

Results

General scheme
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.

Timeline
A calendar of the major parts of the protocol (Fly work a.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Liberase TM	Roche	5401127001	Proteolytic enzyme blend
NaCl	Sigma-Aldrich	S3014
KCl	Sigma-Aldrich	P9541
NaH2PO4	Sigma-Aldrich	S9638
NaHCO3	Sigma-Aldrich	S5761
Glucose	Sigma-Aldrich	G0350500
L-Glutathione	Sigma-Aldrich	G6013
Heat Inactivated Bovine Serum	Sigma-Aldrich	F4135
Insulin Solution	Sigma-Aldrich	I0516
L-Glutamine	Sigma-Aldrich	G7513
Penicillin-Streptomycin Solution	Sigma-Aldrich	P4458
Schneider's Culture Medium	Gibco	21720024
Papain	Worthington	LK003178
2-Mercaptoethanol	Sigma-Aldrich	M6250-100ML
RNeasy Micro Kit	Qiagen	74004	RNA purification kit
RNase-free DNase	Qiagen	79254
SuperScript II Reverse Transcriptase	Life Technologies	18064-014
dNTP Mix	Life Technologies	R0191
MgCl2 Solution	Sigma-Aldrich	M1028-10X1ML
Betaine Solution	Sigma-Aldrich	B0300-1VL
RNaseOUT	Life Technologies	10777-019
Q5 High-Fidelity 2x Master Mix	New England Biolabs	M0492S
MinElute PCR Purification Kit	Qiagen	28004
Nextera XT DNA Library Prepration Kit	Illumina	FC-131-1024
Nextera XT Index Kit	Illumina	FC-131-1001
Test Tube with Cell Strainer Snap Cap	Falcon	352235
Bottle-Top Vacuum Filter Systems	Corning	CLS431153
ThermoMixer F1.5	Eppendorf	5384000020
FACSAria Flow Cytometer	BD Biosciences	656700
HiSeq 2500 Sequencing System	Illumina	SY–401–2501