We are grateful to the members of the Karch lab, to the iGE3 genomics plateform, to the Flow Cytometry core facility of the University of Geneva, and to Dr. Jean-Pierre Aubry-Lachainaye who set the protocol for FACS. We thank Luca Stickley for his help with visualizing the reads on IGV. We thank ourselves for gentle permission to reuse figures, and the editors for prompting us to be creative with writing.
This research was funded by the State of Geneva (CI, RKM, FK), the Swiss National Fund for Research (www.snf.ch) (FK and RKM) and donations from the Claraz Foundation (FK).

1. Drosophila Lines Used and Collection of Males
<ol>
	<li>For this protocol, use males expressing GFP in the SCs and not the main cells. Here, an AbdB-GAL4 driver (described in reference18), recombined with UAS-GFP (on chromosome 2) is used. Other appropriate drivers can also be used. For isolation of SCs under different mutant conditions, cross the mutation into lines containing the SC-specific GFP line.</li>
	<li>Collect two batches of about 25 healthy, virgin males for each genotype and age them at 25 &#176;C for 3-4 days in vials with freshly made Drosophila food. 
	NOTE: As age, mating status, nutrition and social environment each affect reproductive biology, strict protocols must be followed to control for these parameters. Virgin males are aged for 3-4 days after pupal eclosion at 25 &#176;C with a 12 h/12 h light/dark cycle, on standard corn meal-agar-yeast food, in groups of 20-25 males per vial.</li>
</ol>
2. Solutions and Material Preparation
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare Serum Supplemented Medium (SSM): Schneider&#39;s Drosophila medium complemented with 10% heat inactivated fetal bovine serum and 1% Penicillin-Streptomycin.</li>
		<li>Prepare aliquots of 1x trypsin enzyme (e.g., TrypLE Express Enzyme) and store at room temperature.</li>
		<li>Prepare aliquots of papain [50 U/mL] (stored at -20 &#176;C and thawed only once).</li>
		<li>Prepare 1x phosphate buffer saline (PBS, stored at room temperature).</li>
	</ol>
	</li>
	<li>Prepare multiple flame-rounded pipet tips for the physical dissociation of accessory glands. 
	NOTE: Use low retention tips for handling accessory glands as they tend to adhere to untreated plastic. The process of flame-rounding reduces the tip opening and smoothens the edges of the tip. This ensures an efficient dissociation after peptidase digestion without shearing the cell membranes.
	<ol>
		<li>Cut one 200 &#181;L tip with a sharp blade and gently pass it over a flame to round out its tip so that the opening is wider but smooth for handling during step 3.7.</li>
		<li>Narrow the opening of multiple 1,000 &#181;L tips by passing the tip opening near a flame for less than one second. Rotate the tip slightly to avoid over-melting or clogging. Sort the tips made from narrower to wider. This can be done by timing the speed of aspiration. Try to dissociate a small scale sample of AGs to test the efficiency of the narrowest tips. 
		NOTE: These tips are critical for mechanical agitation (steps 5.2 and 5.3). Quality flame-rounded pipet tips allow for complete dissociation while preserving cell viability. As such, these tips are washed thoroughly with water at the end of the procedure for reuse. This allows for day to day reproducible dissociation.</li>
	</ol>
	</li>
</ol>
3. Dissection of Accessory Glands
<ol>
	<li>Put 20 to 25 male Drosophila in a glass dish on ice.</li>
	<li>Dissect 1 male in SSM. Take off its reproductive tract and clear the accessory gland pair from all other tissues apart from the ejaculatory duct. 
	NOTE: Remove testes because the released sperm can create clumps and disturb the dissociation process. Remove the ejaculatory bulb, which makes handling difficult when it floats.</li>
	<li>With forceps, transfer accessory glands to a glass plate filled with SSM at room temperature. Repeat steps 3.2-3.3 20 times to obtain a batch of 20 pairs of glands in SSM. 
	NOTE: These steps will be achieved in 15 to 20 min. AGs should look healthy, and the peristaltic movements of the muscle layer around glands should be visible. GFP can be monitored using a fluorescent microscope.</li>
	<li>Transfer accessory glands to 1x PBS for a 1-2 min wash at room temperature. 
	NOTE: For better dissociation, it is imperative to rinse the glands with PBS. The duration of this step, however, should be limited as the cells show signs of stress in PBS; dissociated secondary cells in PBS rapidly swell and die.</li>
	<li>Dilute 20 &#181;L papain [50 U/mL] into 180 &#181;L of 1x trypsin enzyme to obtain the dissociation solution (to scale up or down keep 9 &#181;L of trypsin enzyme and 1 &#181;L of papain for each male). With forceps, transfer accessory glands to this solution.</li>
	<li>Isolate the tip of accessory glands (containing secondary cells) from the proximal part. Use fine forceps to pinch firmly the middle of a glandular lobe and cut with the sharp tip of a second forceps. Remove the proximal part of accessory glands and the ejaculatory duct to improve dissociation and reduce cell sorting time. 
	NOTE: Dissecting the distal part from 20 pairs of glands will take 15 to 20 min and digestion by peptidases will thus start at room temperature.</li>
	<li>After all gland tips have been dissected, transfer them into a 1.5 mL tube using a special 200 &#181;L pipet tip prepared at Step 2.2.1. It should be wide, rounded and wet prior to handling glands. 
	NOTE: To pipet accessory gland tissue, tips should always be pre-wet with appropriate solution (trypsin enzyme or SSM, keep one tube of each for this purpose). Carefully rinse tips between samples to avoid contamination.</li>
</ol>
4. Tissue Digestion
<ol>
	<li>Place the microtube in a 37 &#176;C shaker for 60 min, rocking at 1,000 rpm. 
	NOTE: Both agitation and digestion time are critical for successful dissociation. Shorter or motionless digestion will give poor dissociation, probably because the outer muscle layer and inner viscous seminal fluid protect accessory gland cells from peptidases.</li>
	<li>Add 1 mL of SSM at room temperature to stop the digestion and proceed immediately to step 5.</li>
</ol>
5. Mechanical Dissociation of the Cells
<ol>
	<li>Using a wide rounded 1,000 &#181;L tip, pre-wet with SSM, transfer the sample to a 24-well plate. Monitor GFP fluorescence under the microscope: most gland tips should look intact and a few SC should be detached.</li>
	<li>Using a rounded narrow pipet tip generated at Step 2.2.2, pipet up and down 3 to 5 times to disrupt the accessory gland tissue. 
	NOTE: Monitor fluorescence to make sure that big tissue patches have been broken up. Repeat step 5.2 if this is not the case.</li>
	<li>Using a very narrow rounded pipet tip (generated at Step 2.2.2), pipet up and down 1-2 times. 
	NOTE: Only individual cells should be visible after Step 5.3. Using 24-well plates is recommended because they enable an easy monitoring of the process. When a few samples were processed and gave satisfactory result (healthy looking secondary cells perfectly dissociated), the pipetting steps will be performed in the microtube.</li>
	<li>Wait at least 15 min to let the cells settle at the bottom of the well and remove the excess SSM to reduce the sorting time. 
	NOTE: Letting cells settle proved superior to alternative methods like centrifugation, and allows a last visual inspection of cells just before FACS.</li>
	<li>Pipet the cells into a clean 1.5 mL tube and pool identical samples (two batches of 20 males for each condition). Rinse wells with SSM to recover last cells, and pipet them into the tube (use a small volume).</li>
	<li>In 1.5 mL microtubes, add 300 &#181;L of Cell Lysis Solution and 1 &#181;L of Proteinase K [50 &#181;g/&#181;L] (solutions provided in the RNA purification kit, see Step 7). Prepare two tubes for each sample (one for MCs and one for SCs).</li>
</ol>
6. FACS (Fluorescence-activated Cell Sorting)
<ol>
	<li>Add viability marker to each sample to be sorted a few minutes before sorting (0.3 mM Draq7). 
	CAUTION: Draq7 should be handled with caution.</li>
	<li>Sort a homogeneous population of live secondary cells (GFP-positive, Draq7-negative cells). In another microtube, sort a population of main cells (smaller, GFP-negative, Draq7-negative cells). Use the following FACS gating strategy: 
	NOTE: Set the cell sorter pressure at 25 PSI and pass cells through a 100 &#181;m nozzle. The sorting rate is around 2,000 cells/s.
	<ol>
		<li>Exclude debris and select total cells based on FSC-SSC (Figure 2E) in order to exclude debris.</li>
		<li>Exclude dead cells. Excite Draq7 with a 640 nm laser and collect emitted fluorescence with a 795/70 band-pass filter. Gate Draq7-positive cells out (Figure 2F).</li>
		<li>Remove doublets using a double gating on SSC-H vs SSC-W and FSC-A vs FSC-H (Figure 2H). 
		NOTE: Exclude cell doublets stringently using double gating, to reduce main cell contamination.</li>
		<li>Sort ~550 GFP-positive cells into a 1.5 mL microtube with Lysis buffer and Proteinase K. This population is considered secondary cells (SC, Figure 2G). Excite GFP at 488 nm and collect emitted fluorescence with a 526/52 band-pass filter.</li>
		<li>Sort ~1,000 GFP-negative cells, homogeneous and small in size, into a 1.5 mL microtube with Lysis buffer and Proteinase K. This population is considered main cells (MC, Figure 2G).</li>
		<li>Vortex all samples. 
		NOTE: From 40 males (~40 x 80 SC = 3,200 secondary cells), 600 to 800 live singlet secondary cells are obtained (around 20-25% retrieval). Stop sorting around 550 SC and 1,000 MC to normalize samples.</li>
	</ol>
	</li>
</ol>
7. RNA Extraction
NOTE: We used Epicentre MasterPure RNA Purification Kit for RNA extraction, with the following adaptations. Other kits might be used as long as the yield is high enough to prepare a library for sequencing from ~500 cells (2 ng RNAs was used here).
<ol>
	<li>Heat samples at 65 &#176;C for 15 min and vortex every 5 min to complete cell lysis.</li>
	<li>Place samples on ice for 5 min. Follow manufacturer&#39;s recommendations for nucleic acids precipitations (parts &#34;Precipitation of Total Nucleic Acids&#34; and &#34;Removal of Contaminating DNA from Total Nucleic Acid Preparations&#34;).</li>
	<li>Suspend RNA pellet in 10 &#181;L of RNase-free TE buffer.</li>
	<li>Add RNase inhibitor (optional).</li>
	<li>Store samples at -80 &#176;C.</li>
</ol>
8. Quality Controls of RNA Quantity, Quality and Specificity
<ol>
	<li>Estimate RNA quality and concentration. Due to the small volume and concentration, use RNA 6000 Pico chips here. Good quality RNA is defined as non-degraded, visible as a low baseline with sharp peaks corresponding to rRNAs.</li>
	<li>RT-qPCR to control the identity of sorted cells
	<ol>
		<li>Perform reverse transcription with 2 ng of total RNAs using random hexamers as primers. Perform RT on secondary cell RNA and main cell RNA. 
		NOTE: cDNAs can be diluted, aliquoted, and kept frozen for later use.</li>
		<li>Perform real time quantitative PCR using appropriate primer pairs to quantify housekeeping genes (alpha-Tubulin, 18S rRNA), secondary cell specific genes (MSA, Rab19, Abd-B) and main cell specific gene (Sex peptide).</li>
	</ol>
	</li>
</ol>
9. Sequencing (cDNA Library Preparation, Sequencing and Data Analysis)
<ol>
	<li>Use 2 ng of total RNAs to synthesize cDNAs with polydT primers. Use SMARTer technology to amplify them for amplification.</li>
	<li>Use a Nextera XT kit to prepare the library.</li>
	<li>Sequence using multiplexed, 100 nucleotides single reads sequencing (even though 50 nucleotides reads are appropriate for most purposes) to yield around 30 million reads for each sample.</li>
</ol>
10. Data Analysis
<ol>
	<li>Run FastQC.</li>
	<li>Use the STAR aligner to map the reads on the UCSC dm6 Drosophila reference genome and generate .bam files. Use Integrative Genomics Viewer (IGV) to visualize reads on the genome browser.</li>
	<li>Use HTSeq to perform gene counts.</li>
	<li>Use the Trimmed Mean of M-values (TMM) method to normalize gene counts22. Use edgeR to perform statistical analysis of differential expression, MA plots and PCA.</li>
	<li>For gene expression analysis and statistics, use General Linear Model, quasi-likelihood F-test with False Detection Rate (FDR) and Benjamini &#38; Hochberg correction (BH).</li>
</ol>

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The authors have no disclosure.

Methods for cell dissociation from Drosophila tissue like imaginal discs are already described23. Our attempts to simply use these procedures on accessory glands failed, encouraging us to develop this new protocol. Protease digestion and mechanical trituration were critical steps we troubleshot for the success of the procedure, and we thus placed many notes in sections 2 to 5 to help experimenters to obtain satisfactory results. For dissociation to be successful, peptidases must reach the...

Organs are made up of multiple cell types, each with discrete functions and sometimes expressing vastly different sets of genes. To get a precise understanding of how an organ functions, it is often critical to study each distinct cell type that makes up this organ. One of the primary methods used to explore possible function is transcriptome analysis. This powerful method provides a snapshot of the gene expression in a cell, to reveal active processes and pathways. However, this type of analysis is often difficult for rare cell populations that must be purified from far more-abundant neighboring cells. For example, the Drosophila male accessory gland is an organ made up primarily of two secretory cell types. As the rarer of the two cell types comprises only 4% of the cells of this gland, the use of a cell-type specific transcriptome analysis had not been used to help determine the function of these cells.
Accessory glands (AGs) are organs of the male reproductive tract in insects. They are responsible for the production of most of the proteins of the seminal fluid (seminal fluid proteins (SFPs) and accessory gland proteins (ACPs)). Some of these SFPs are known to induce the physiological and behavioral responses in mated females, commonly called the post-mating response (PMR). Some of the PMRs include: an increased ovulation and egg-laying rate, the storage and release of sperm, a change in female diet, and a decrease in female receptivity to secondary courting males1,2. As insects impact many major societal issues from human health (as vectors for deadly diseases) to agriculture (insects can be pests, yet are critical for pollination and soil quality), understanding insect reproduction is an important area of research. The study of AGs and ACPs has been advanced significantly with the model organism Drosophila melanogaster. These studies have highlighted the role of AGs and some of the individual proteins that they produce in creating the PMR, impacting the work in other species like the disease vector Aedes aegypti3,4, and other insects1,5. Furthermore, as AGs secrete the constituents of the seminal fluid1,6 they are often thought of as the functional analog of the mammalian prostate gland and seminal vesicle. This function similarity combined with molecular similarities between the two tissue-types, have made the AGs a model for the prostate gland in flies7.
Within the Drosophila male, there are two lobes of accessory glands. Each lobe can be seen as a sac-like structure made up of a monolayer of secretory cells surrounding a central lumen, and wrapped by smooth muscles. As mentioned above, there are two morphologically, developmentally and functionally distinct secretory cell types making up this gland: the polygonal-shaped main cells (making up ~96% of the cells), and the larger, round secondary cells (SC) (making up the remaining 4% of cells, or about 40 cells per lobe). It has been shown that both cell types produce distinct sets of ACPs to induce and maintain the PMR. Most of the data obtained to date highlight the role of a single protein in triggering most of the characteristic behaviors of the PMR. This protein, the sex peptide, is a small, 36 amino acid peptide that is secreted by the main cells8,9,10. Although the sex peptide seems to play a major role in the PMR, other ACPs, produced by both the main and secondary cells, have also been shown to affect various aspects of the PMR11,12,13,14,15,16,17. For example, based on our current knowledge, the SCs, via the proteins they produce, seem to be required for the perpetuation of the SP signaling after the first day18.
Given the rarity of the SCs (only 80 cells per male), all our knowledge about these cells and the proteins they produce comes from genetics and candidate approaches. Thus far, only a relatively small list of genes has been shown to be SC-specific. This list includes the homeodomain protein Defective proventriculus (Dve)19, the lncRNA MSA20, Rab6, 7, 11 and 1921, CG1656 and CG1757511,15,21 and the homeobox transcription factor Abdominal-B (Abd-B)18. Previously, we have shown that a mutant deficient for both the expression of Abd-B and the lncRNA MSA in secondary cells (iab-6cocuD1 mutant) shortens the length of the PMR from ~10 days to only one day12,18,20. This phenotype seems to be caused by the improper storage of SP in the female reproductive tract12,18,20. At the cellular level, the secondary cells of this mutant show abnormal morphology, losing their characteristic vacuole-like structures18,20,21. Using this mutant line, we previously attempted to identify genes involved in SC function by comparing the transcriptional profiles of whole AGs from either wild type or mutant accessory glands12. Also, other labs showed that SC number, morphology and vacuolar content depend on male diet, mating status and age21,25,26.
Although positive progress was made using these approaches, a full SC transcriptome was far from achieved. The rareness of these cells in this organ made it difficult to progress further even from wild type cells. For testing gene expression, changes in these cells after particular environmental stimuli would be even harder. Thus, a method for isolating and purifying SC RNA that was fast and simple enough to perform under different genetic backgrounds and environmental conditions was needed.
Both the Abd-B and MSA genes require a specific 1.1 kb enhancer from the Drosophila Bithorax Complex (called the D1 enhancer) for their expression in SCs18,20. This enhancer has previously been used to create a GAL4 driver that, when associated with a UAS-GFP, is able to drive strong GFP expression specifically in SCs. Thus, we used this line as the basis for a FACS protocol to isolate these cells from both wild type and iab-6cocuD1 AGs). As iab-6cocuD1 mutant SCs display a different cellular morphology, we show that this protocol can be used to isolate cells for the determination of their transcriptome from this rare cell type under vastly different conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-wells Tissue Culture plate</td>
			<td>VWR</td>
			<td>734-2325</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope for dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular with light source for GFP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Draq7 0.3 mM</td>
			<td>BioStatus</td>
			<td>DR71000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic microtubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>Beckman Coulter</td>
			<td>MoFlo Astrios</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine dissection forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>heat inactivated prior to use</td>
		</tr>
		<tr>
			<td>Glass dishes for dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImProm-II Reverse Transcription System</td>
			<td>Promega</td>
			<td>A3800</td>
			<td></td>
		</tr>
		<tr>
			<td>MasterPure RNA Purification Kit</td>
			<td>Epicentre</td>
			<td>MCR85102</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT kit</td>
			<td>illumina</td>
			<td></td>
			<td><a href="https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html">https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html</a></td>
		</tr>
		<tr>
			<td>P1000</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P20</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P200</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td></td>
			<td></td>
			<td>50U/mL stock</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>home made</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>PolydT primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Random hexamer primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico kit</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila medium</td>
			<td>Gibco</td>
			<td>21720-001</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer cDNA synthesis kit</td>
			<td>Takara</td>
			<td></td>
			<td><a href="https://www.takarabio.com/products/cdna-synthesis/cdna-synthesis-kits/smarter-cdna-synthesis-kits">https://www.takarabio.com/products/cdna-synthesis/cdna-synthesis-kits/smarter-cdna-synthesis-kits</a></td>
		</tr>
		<tr>
			<td>SYBR select Master mix for CFX</td>
			<td>applied biosystems</td>
			<td>4472942</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker</td>
			<td>Hangzhou Allsheng intruments</td>
			<td>MS-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tipone 1250 &#956;l graduated tip</td>
			<td>Starlab</td>
			<td>S1161-1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Tipone 200 &#956;l bevelled tip</td>
			<td>Starlab</td>
			<td>S1161-1800</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol presented here enables one experimenter to isolate secondary cells from Drosophila male accessory glands and to extract their RNA in the course of one single day (Figure 1).
We use the Abd-B-GAL4 construct18 to label secondary cells (SC) but not main cells (MC) with GFP (Figure 2A). One objective of...

Watch this Scientific Journal Video about A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands at JoVE.com

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.

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 ...

a-facs-based-protocol-to-isolate-rna-from-secondary-cells-drosophila

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JoVE Journal

Genetics

6.9K Views.  University of Geneva. 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.

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

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Purification of Low-abundant Cells in the Drosophila Visual System

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.

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 ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

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

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Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

Purification of Low-abundant Cells in the Drosophila Visual System

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Use of Drosophila S2 Cells for Live Imaging of Cell Division