We thank Janice Fischer for providing the UAS-EGFP::Msp-300KASH plasmid. The mAB24B10 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology. The repo-GAL4 stock (BL7415) was obtained from the Bloomington Stock Center at Indiana University. Support from the American Cancer Society Institutional Research Grant (IRG #58-006-53) to the Purdue University Center for Cancer Research is gratefully acknowledged. Jingqun Ma is supported by an Agricultural Research at Purdue Assistantship in Food and Agriculture from Purdue University.

1. Generation and Characterization of EGFP::KASH Drosophila
<ol>
	<li>Cross Gal4 driver flies to UAS-EGFP::Msp-300KASH flies. Alternatively, recombinant flies that carry both the Gal4 driver and the UAS-EGFP::Msp-300KASH transgene can be generated using standard genetic techniques. This second approach is advantageous if large numbers of progeny are required.</li>
	<li>Characterize expression of the EGFP::KASH marker using standard microscopy techniques. Note: EGFP expression can be observed by standard microscopy techniques in dissected, fixed Drosophila tissues, or in whole larvae, and does not require use of an antibody.
	<ol>
		<li>Determine the expression pattern of the EGFP::KASH marker in the entire organism under a fluorescence dissecting microscope (see Materials PDF). Note: Many Gal4 drivers, including the repo-GAL4 driver shown in Figure 1, exhibit nonspecific patterns of expression in other larval tissues such as the salivary gland (see Results).</li>
		<li>Analyze the EGFP::KASH expression pattern in the dissected tissue of interest using confocal microscopy.
		<ol>
			<li>Fix the tissue of interest using formaldehyde at a final concentration of 4%, and stain DNA using DAPI at a final concentration of 0.1 &#956;g/ml to visualize the localization of the EGFP::KASH tag relative to DNA.</li>
			<li>Acquire images using either a&#160;40X / 1.30 oil immersion objective or a&#160;20X / 0.75 oil immersion objective, depending on the size of the tissue of interest. The following excitation/emission conditions can be used for image acquisition: 408 nm/425-475 nm (DAPI); 488 nm /525-550 nm (GFP).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Isolate Nuclei from Drosophila Larval Tissues
<ol>
	<li>Prepare equipment for the larval tissue dissection and subsequent nuclei isolation. Note that whole dechorionated embryos could also be used as starting material if desired. It is recommended to use partially dissected larval tissues rather than entire larvae if the cell type of interest is present in a specific tissue such as the imaginal disc. This reduces nonspecific binding, and also eliminates problems that can be caused by expression of some Gal4 drivers in nontarget cells.
	<ol>
		<li>Prepare two pairs of sharp forceps (see Materials PDF), one siliconized 9-well glass plate (see Materials PDF), and PBT (1x PBS, pH 7.4; 0.1% Tween-20).</li>
		<li>Prebind the GFP antibody to the magnetic beads. This step should be started before beginning the dissection. Add 10 &#956;l of the magnetic beads (Invitrogen, see Materials PDF) and 2 &#956;g of GFP antibody (Roche, see Materials PDF) to 400 &#956;l of wash buffer (1x PBS, pH 7.4; 2.5 mM MgCl2) in a 1.5 ml tube. The amount of beads and antibody used can be optimized if necessary based on the amount of target in the sample and the binding affinity of the antibody to the beads.</li>
		<li>Incubate the beads and antibody with rotation for at least 30 min at room temperature.</li>
		<li>Place the 1.5 ml tube containing the beads and antibody in the magnetic rack (see Materials PDF) and allow the beads to bind to the magnetic for approximately 1-2 min. Remove the supernatant containing the unbound antibody and wash buffer using a 1 ml pipette. The beads will remain bound to the wall of the 1.5 ml tube on the side adjacent to the magnet.</li>
		<li>Rinse the beads twice with 1 ml of wash buffer each time as described in section 2.1.4. Remove the 1.5 ml tube from the magnetic rack and invert briefly to mix the beads and wash buffer during each wash step.</li>
		<li>Store the antibody-bound beads in wash buffer until ready to continue with step 2.7. The beads should not be allowed to dry out at any stage of the protocol.</li>
	</ol>
	</li>
	<li>Dissect larval tissues in PBT and transfer dissected tissues to one well of the 9-well dish. Typically, dissection of the optic lobe and eye imaginal disc from 100 third instar larvae provides sufficient material for downstream gene expression analysis following nuclei isolation.</li>
	<li>Rinse the isolated tissue in nuclear extraction buffer (10 mM HEPES-KOH, pH 7.5; 2.5 mM MgCl2; 10 mM KCl) in a 1.5 ml tube. Transfer isolated larval tissues to a 1 ml Dounce Homogenizer (see Materials PDF) on ice that contains 1 ml of fresh nuclear extraction buffer. Incubate on ice for 5 min. It is not necessary to add protease inhibitors if RNA is to be extracted following nuclei isolation.</li>
	<li>Disrupt the tissue by 20 strokes with the loose pestle and then incubate the sample on ice for 10 min to allow the cells to swell. Extract the nuclei from the cells using 15 strokes with the tight pestle. The number of strokes with the loose and tight pestles needs to be optimized for different tissues and cell types; excess homogenization can result in sheared nuclei, while insufficient homogenization will not extract all nuclei from the tissue.</li>
	<li>Filter the homogenate, which contains the nuclei and cellular debris, through a 40 &#956;m pore-size strainer (see Materials PDF) that has been prerinsed with nuclear homogenization buffer. Collect the filtered homogenate in a fresh tube.</li>
	<li>Collect pre-isolation samples for subsequent analysis to determine nuclei yield, nuclei integrity, and to determine transcript levels of target genes prior to nuclei isolation.
	<ol>
		<li>Transfer 50 &#956;l of the filtered homogenate to a fresh 1.5 ml tube using a pipette. This is the pre-isolation sample. Add 500 &#956;l of Trizol reagent (see Materials PDF, CAUTION), invert to mix, and store at -20 &#176;C for subsequent RNA isolation (step 3.1).</li>
		<li>Transfer 20 &#956;l of the filtered homogenate to a fresh 1.5 ml tube.
		<ol>
			<li>Add DAPI to a final concentration of 0.1 &#956;g/ml, and incubate sample on ice for 10 min.</li>
			<li>Transfer DAPI-stained nuclei to a poly-lysine coated coverslip, and place on a microscope slide. Seal the coverslip using clear nail polish.</li>
			<li>Check the nuclei integrity, and presence of GFP-tagged nuclei of interest using fluorescent microscope. Note: It is not necessary to fix the nuclei, or to stain for GFP if these slides are analyzed reasonably quickly following preparation (within 24 hr).</li>
		</ol>
		</li>
		<li>Transfer 10 &#956;l of the filtered homogenate to a fresh 1.5 ml tube and add 90 &#956;l of wash buffer (1x PBS, pH 7.4; 2.5 mM MgCl2). Add DAPI to a final concentration of 0.1 &#956;g/ml, and incubate on ice for 10 min. Determine the nuclei yield in the pre-isolation sample using a hemocytometer to count the DAPI-positive nuclei using epifluorescence under a 10X air objective.</li>
	</ol>
	</li>
	<li>Place the 1.5 ml tube containing the antibody-bound beads (prepared in section 2.1.6) in a magnetic rack, and allow the magnetic beads to bind to the magnet for at least 2 min as described in section 2.1.4. Remove the wash buffer from the antibody-bound beads using a 1 ml pipette, and add the filtered homogenate from step 2.5 to the 1.5 ml tube using a 1 ml pipette.</li>
	<li>Gently invert the tube several times to mix, and incubate at 4 &#176;C for 30 min with end-over-end agitation. Avoid bubbles in the tube if possible since these can result in clumping of the nuclei.</li>
	<li>Place the 1.5 ml tube containing the antibody-bound beads and homogenate in a magnetic rack, and allow the magnetic beads to bind to the magnet for at least 2 min as described in section 2.1.4. Remove the homogenate containing the unbound nuclei fraction using a 1 ml pipette.</li>
	<li>Wash the sample 3x with 1 ml of wash buffer each time. Remove the 1.5 ml tube containing the beads, bound nuclei and wash buffer from the rack and invert to mix several times, then place the tube in the magnetic rack as described in step 2.9 and remove the supernatant using a 1 ml pipette.</li>
	<li>Add 150 &#956;l of wash buffer to the beads, which should be bound to the target nuclei. This is the post-isolation sample. Prepare samples for microscopy analysis and subsequent RNA extraction.
	<ol>
		<li>Transfer 20 &#956;l of the post-isolation sample to a fresh 1.5 ml tube and stain with DAPI and analyze as described in section 2.6.2. Count the GFP+/DAPI+ and GFP-/DAPI+ nuclei captured by magnetic beads to determine the purity of the enriched GFP+ nuclei in the post-isolation sample.</li>
		<li>Add 1 ml of Trizol reagent to the remainder of the post-isolation sample, invert to mix, and store at -20 &#176;C for subsequent RNA isolation (step 3.1). Note: Samples in Trizol can be stored for several weeks if necessary at -20 &#176;C. It is not necessary to elute the nuclei from the magnetic beads prior to RNA extraction using Trizol reagent, because the beads do not interfere with the subsequent RNA isolation.</li>
	</ol>
	</li>
</ol>
3. Determine Transcript Levels in Nuclei Isolated from Larval Tissues
<ol>
	<li>Isolate RNA from the pre-isolation and post-isolation samples prepared in sections 2.6.1 and 2.11.2 using the standard protocol described for Trizol-based RNA extraction (see Materials PDF) with the following modifications:
	<ol>
		<li>Following precipitation of the RNA pellet, add 19.2 &#956;l of RNase-free water and 0.8 &#956;l of RNASecure reagent (see Materials PDF) to the pellet and incubate at 60 &#176;C for 20 min to inactivate RNAses.</li>
	</ol>
	</li>
	<li>Determine the RNA concentration with a fluorescent-RNA binding dye such as the Qubit RNA assay kit (see Materials PDF) using the QubitR 2.0 Fluorometer following the manufacturer&#8217;s protocol.</li>
	<li>Synthesize cDNA using an amplifying reverse transcriptase such as the EpiScript Reverse Transcriptase kit and oligodT primers following the manufacturer&#8217;s instructions. Note: Typically, 50 to 100 ng of RNA generates sufficient cDNA to perform multiple gene expression analyses. Equivalent amounts of pre-isolation and post-isolation RNA should be used to generate cDNA.</li>
	<li>Add 180 &#956;l of nuclease-free water to the 20 &#956;l cDNA sample to dilute this 10-fold prior to real-time quantitative PCR (qPCR) analysis. This dilution is an important step, because undiluted cDNA samples can inhibit the qPCR.</li>
	<li>Prepare a qPCR master mix with polymerase and dNTPs, 4 pmoles each of forward and reverse primer, made up with sterile water to the final reaction volume of 20 &#956;l.
	<ol>
		<li>Design qPCR primers to target genes that are expected to be expressed in the cell type of interest, and at the developmental stage at which the tissue is collected. Design primers to span an intron if possible, so that genomic and cDNA PCR products can be distinguished. Note: If the gene expression profile of the target cell type is not known, primers against eGFP, which should be expressed specifically in the tagged nuclei population, can be used to optimize the protocol for a particular cell type and tissue (Table 1).</li>
	</ol>
	</li>
	<li>Determine the relative transcript levels of each gene of interest in the pre-isolation and post-isolation samples by comparison to a dilution series of cDNA prepared from the whole tissue. Transcript levels of target genes should be normalized to a reference gene such as Rpl32 (or preferably several reference genes).</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+klarsicht+has+distinct+subcellular+localization+domains+for+nuclear+envelope+and+microtubule+localization+in+the+eye.">Drosophila klarsicht has distinct subcellular localization domains for nuclear envelope and microtubule localization in the eye.</a> Genetics. 168, 1385-1393 (2004).</li><li>Patterson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+Klarsicht+and+nuclear+lamin+in+developmentally+regulated+nuclear+migrations+of+photoreceptor+cells+in+the+Drosophila+eye.">The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye.</a> Mol. Biol. 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The authors have nothing to disclose.

This protocol can be used to isolate or highly enrich specifically tagged nuclei from mixed cell populations in Drosophila embryonic or larval tissues. Using this protocol, nuclei can be isolated from dissected tissues in approximately 1 hr. The purity and yield of the isolated nuclei must be experimentally determined for each cell type. It can be difficult to quantify the bead-bound nuclei at the post-isolation stage of the protocol using a hemocytometer because of the beads present in the sample. Thus, if exac...

Drosophila tissues such as the central nervous system contain a complex mixture of cell types. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it is first necessary to isolate a homogenous population of specific cells in sufficient quantities to enable downstream applications. Methods to isolate cells from intact tissues include laser microdissection, and fluorescence-activated cell sorting (FACS) of whole cells. While FACS has been used to isolate cells and nuclei from Drosophila embryos and from Caenorhabditis elegans for gene expression and chromatin profiling1-3, FACS and laser microdissection can be difficult to perform successfully in tissues that contain highly intermixed cell types or that contain cells with irregular morphology, such as neurons. To overcome this difficulty, nuclei rather than cells can be isolated from specific cell types and used for subsequent gene expression profiling. Importantly, microarray-based mRNA expression analysis using nuclear RNA samples shows generally comparable results with that performed using total RNA4,5. Moreover, gene expression analysis using nuclear RNA has been successfully used to study gene expression in multiple organisms including C. elegans, Arabidopsis thaliana, Drosophila, and humans4,52,3.
Several approaches have recently been described for the isolation of specific populations of labeled nuclei from Drosophila tissues that are suitable for gene expression analysis and/or chromatin immunoprecipitation. The batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) method utilizes FACS to isolate fixed nuclei on the basis of cell-type specific expression of nuclear-localized GFP2. This approach has been successfully used to analyze the distribution of histone modifications and transcription factors using chromatin immunoprecipitation of isolated nuclei from the mesoderm of Drosophila embryos2. However, FACS-based approaches may be less suitable for isolating labeled nuclei that constitute only a small proportion of a mixed population due to the increased sort time needed to obtain suitable numbers for downstream applications. To overcome these limitations, several groups have utilized affinity-based isolation techniques to purify nuclei that are labeled with a specific epitope in a particular cell type. The isolation of nuclei tagged in specific cell types (INTACT) method developed for use in Arabidopsis thaliana6,7 has recently been adapted for use in Drosophila8. In this method, a nuclear envelope fusion protein that is a substrate for in vivo biotinylation is coexpressed with the Escherichia coli biotin ligase BirA in specific cell types. Biotin-labeled nuclei can be subsequently purified from mixed populations using streptavidin-based affinity isolation. Using this approach, nuclei were successfully labeled and isolated from the mesoderm of Drosophila embryos in which a nuclear envelope fusion protein was expressed under control of a mesoderm-specific enhancer8. The authors also generated nuclear envelope fusion proteins that can be expressed in any cell type under control of the Gal4 regulatory sequence, UAS9. This approach is capable of rapidly isolating subsets of labeled nuclei from mixed populations, but requires three separate transgenic constructs and may therefore be unsuitable for particular genetic applications. Recently, an approach has been described in which SUN (Sad1 and UNC-84) domain-containing proteins that localize to the inner membrane of the nuclear envelope were tagged with fluorescent proteins and expressed under control of the Gal4/UAS system10. Nuclei were isolated in the presence of nonionic detergent to remove the outer membrane of the nuclear envelope, and affinity-purified using magnetic beads coupled to anti-GFP antibodies. This approach was successfully used to isolate small populations of labeled nuclei from specific neuronal subtypes within the adult brain of Drosophila10.
Here, a protocol for isolation of labeled nuclei from a mixed population of cells from Drosophila larval tissue is described. This method was developed independently, but is similar to the approach described by Henry et al.10&#160;First, nuclei were labeled with a fluorescent tag that is expressed only in specific cell types in Drosophila melanogaster to facilitate the subsequent isolation of tagged nuclei from mixed populations using an affinity-based approach. To label nuclei, the KASH (Klarsicht/Anc-1/Syne homology) domain was utilized. The KASH domain is a transmembrane domain that localizes to the outer membrane of the nuclear envelope, in part through interactions with SUN domain-containing proteins within the perinuclear space11. The C-terminal KASH domain of proteins such as Drosophila Msp-300 and Klarsicht anchors these proteins to the outer nuclear membrane, while their N-terminal domains interact with cytoskeleton proteins such as actin or microtubules in the cytoplasm12-14. Constructs were generated in which the KASH domain of Drosophila Msp-300 was fused to the C-terminus of EGFP, under control of the Gal4 regulatory sequence, UAS in the pUAST-attB vector15,16. Using phiC31 site-specific integration, transgenic flies were generated in which the UAS-EGFP::Msp-300KASH transgene was inserted in the attP2 loci on chromosome 3L17. The UAS-EGFP::Msp-300KASH flies can be crossed with flies that express the Gal4 driver in a particular cell type, resulting in the targeted expression of EGFP on the outer nuclear membrane in the cell type of interest. Labeled nuclei can then be purified from mixed cell populations using antibodies against GFP coupled to magnetic beads. In this approach, the use of nonionic detergent is not required because the EGFP tag is localized to the cytoplasmic side of the outer nuclear membrane, and is therefore accessible to antibodies.
The protocol described below can be used to isolate/enrich EGFP-labeled nuclei from specific cell types in Drosophila larval tissues, to quantify the purity and yield of isolated nuclei, and to extract nuclear RNA suitable for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) gene expression analysis. The isolation and affinity-purification of nuclei (excluding tissue dissection) can be performed in less than one hour. Results are presented showing that glial nuclei can be successfully isolated from the larval optic lobe and eye imaginal disc, and used for subsequent gene expression analysis. It is anticipated that this approach will be useful for the isolation/enrichment of labeled nuclei from embryonic and larval tissues in which the target cells constitute less than 5% of the overall population. All Drosophila stocks and plasmids generated in this study are available from the authors upon request.

<table><tbody><tr><td>Anti-chaoptin antibody (mouse, monoclonal)</td><td>Developmental Studies Hybridoma Bank</td><td>mAB24B10</td><td></td></tr><tr><td>Anti-GFP antibody (mouse, monoclonal)</td><td>Roche</td><td>11814460001</td><td>This specific antibody is recommended for this protocol. However, alternative antibodies could also be used.</td></tr><tr><td>2x Bullseye EvaGreen qPCR Mastermix with ROX</td><td>MidSci</td><td>BEQPCR-R</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole, dihydrochloride salt)</td><td>Biotium</td><td>40011</td><td></td></tr><tr><td>Dounce Homogenizer (1 ml)</td><td>VWR</td><td>62400-595</td><td></td></tr><tr><td>Dumont #5 Tweezers, 11 cm</td><td>World Precision Instruments</td><td>14095</td><td></td></tr><tr><td>Dynabeads Protein G for Immunoprecipitation</td><td>Invitrogen</td><td>10003D</td><td>This specific brand of magnetic beads is recommended for this protocol.</td></tr><tr><td>EpiScript Reverse Transcriptase</td><td>Epicentre</td><td>ERT12910K</td><td>&lt;a href=&quot;http://www.epibio.com/docs/default-source/protocols/episcript-reverse-transcriptase.pdf&quot;&gt;http://www.epibio.com/docs/default-source/protocols/episcript-reverse-transcriptase.pdf&lt;/a&gt;</td></tr><tr><td>Falcon cell strainers, 40 &amp;mu;m pore size</td><td>VWR</td><td>21008-949</td><td>Alternative pore sizes could be used, depending on the size of the target cell nuclei.</td></tr><tr><td>Magnetic stand (8 x 1.5 ml tubes)</td><td>Millipore</td><td>LSKMAGS08&amp;nbsp;</td><td></td></tr><tr><td>Mini tube rotator</td><td>Genemate</td><td>H-6700</td><td></td></tr><tr><td>Qubit starter kit</td><td>Life Technologies</td><td>Q32871</td><td>Similar fluorescence-based methods for quantifying RNA yield could be used. &lt;a href=&quot;http://tools.invitrogen.com/content/sfs/manuals/mp32852.pdf&quot;&gt;http://tools.invitrogen.com/content/sfs/manuals/mp32852.pdfZ&lt;/a&gt;</td></tr><tr><td>RNAsecure (Ambion)</td><td>Life Technologies</td><td>AM7010</td><td>The use of this reagent to inactivate RNAses is optional.</td></tr><tr><td>RT-PCR machine Thermal Cycler</td><td>BioRad</td><td>CFX Connect Thermal Cycler</td><td></td></tr><tr><td>Siliconized 9-well glass plate</td><td>Hampton Research</td><td>HR3-134</td><td></td></tr><tr><td>Stereomicroscope Fluorescence Adapter (Royal Blue (440-460 nm) excitation + yellow barrier</td><td>Nightsea</td><td></td><td>This adaptor enables GFP fluorescence to be examined using a typical dissecting microscope (eg Nikon)</td></tr><tr><td>StereoZoom Microscope 83604 Set with 10X Wide Field Eyepieces and Universal Boom Stand</td><td>Nikon</td><td>SMZ 745</td><td></td></tr><tr><td>Thermal Cycler</td><td>BioRad</td><td>T100</td><td></td></tr><tr><td>Trizol reagent</td><td>Life Technologies</td><td>15596018</td><td>CAUTION; Alternative RNA extraction methods can be used. &lt;a href=&quot;http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf&quot;&gt;http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf&lt;/a&gt;</td></tr><tr><td>Tween 20</td><td>Amresco</td><td>0777-1L</td><td></td></tr></tbody></table>

affinity-based isolation of tagged nuclei from drosophila tissues for gene expression analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

The repo-GAL4 driver (Bloomington stock number 7415) is specifically expressed in glial cells in the Drosophila nervous system at multiple stages of development18. Flies were generated that stably express UAS-EGFP::Msp-300KASH under control of the repo-GAL4 driver using standard genetic techniques. To characterize the expression pattern of the EGFP::KASH transgene in these flies, the pattern of GFP expression in the dissected optic lobe and eye imaginal disc from ...

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Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

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14.7K Views.  Purdue University. Drosophila tissues often contain a heterogeneous mixture of cell types. To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Video: Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Developmental Biology

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

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High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands