Regional Cell Specific RNA Expression Profiling of FACS Isolated Drosophila Intestinal Cell Populations

Devanjali Dutta1, Nicolas Buchon2, Jinyi Xiang1, Bruce A. Edgar1

1 DKFZ‐ZMBH Alliance, University of Heidelberg, Heidelberg, 2 Department of Entomology, Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2F.2
DOI:  10.1002/9780470151808.sc02f02s34
Online Posting Date:  August, 2015
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The adult Drosophila midgut is built of five distinct cell types, including stem cells, enteroblasts, enterocytes, enteroendocrine cells, and visceral muscles, and is divided into five major regions (R1 to R5), which are morphologically and functionally distinct from each other. This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type–specific transcriptome profiling from the five different regions. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4‐UAS bipartite system and fluorescent‐activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells of each region, and linear RNA amplification is used to obtain sufficient amounts of high‐quality RNA for analysis by microarray, RT‐PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types in the different regions under normal and various experimental conditions. © 2015 by John Wiley & Sons, Inc.

Keywords: Drosophila intestinal stem cells; fluorescent‐activated cell sorting (FACS); cell type–specific RNA isolation; transcriptome profiling/RNASeq; enterocyte

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Purification of Drosophila Intestinal Cells by Fluorescent‐Activated Cell Sorting (FACS)
  • Basic Protocol 2: RNA Isolation and Amplification
  • Basic Protocol 3: Immunofluorescence Staining of Sorted Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation and Purification of Drosophila Intestinal Cells by Fluorescent‐Activated Cell Sorting (FACS)

  • Transgenic adult fly Gal4 lines driving GFP or YFP expression: esg‐Gal4 (ISCs +EBs), Dl‐Gal4 (ISCs), Su(H)‐Gal4 (EBs), Myo1A‐Gal4 (ECs), How‐Gal4 (visceral muscle) and Rab3‐YFP or Prosv1‐Gal4 (EEs) and UAS‐mcd8‐GFP/UAS‐nls‐GFP
  • 10 × PBS‐DEPC (see recipe)
  • RNaseZap (Ambion) or 70% ethanol
  • Elastase (see recipe) or collagenase type IV (see recipe)
  • 1 mg/ml propidium iodide
  • 10% bleach
  • DEPC‐treated water (see recipe)
  • Wild‐type control wIII8 flies
  • Dissection dishes
  • Dissecting tools: scissors, fine forceps
  • Dissecting microscope
  • 1.5‐ml microcentrifuge tubes
  • 27°C heating block with shaking
  • Refrigerated microcentrifuge
  • 25‐ and 70‐μm filter units (BD Biosciences)
  • FACSAria II cell sorter (Becton Dickinson)
  • FACS tubes with filter tops (BD Biosciences)
  • BD FACSDiva v6.1.1 or similar software

Basic Protocol 2: RNA Isolation and Amplification

  • Sorted cells (see protocol 1)
  • Arcturus PicoPure RNA isolation kit (Applied Biosystems)
  • 70% RNase‐free ethanol
  • Arcturus RiboAmp HS PLUS RNA amplification kit (Applied Biosystems)
  • SuperScript III reverse transcriptase (Invitrogen, cat. no. 18080‐44)
  • 1.5‐ml microcentrifuge tubes
  • Parafilm
  • 42°C water bath
  • Refrigerated microcentrifuge
  • Thermal cycler
  • Qubit 2.0 fluorometer (Invitrogen)

Basic Protocol 3: Immunofluorescence Staining of Sorted Stem Cells

  • Sorted cells (see protocol 1)
  • Schneider's medium (Prom°Cell)
  • Fetal bovine serum (FBS)
  • 16% w/v formaldehyde (Alfa Aesar)
  • 0.15% PBST (see recipe)
  • 5% NGS in PBST (see recipe)
  • Antibodies: mouse monoclonal anti‐Delta (1:100), rabbit polyclonal anti‐GFP (1:1000) (Invitrogen)
  • 0.1% DAPI
  • Vectashield mounting medium (Vector)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Positively charged slides
  • Petri plates
  • Coverslips
  • Microscope
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Literature Cited

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