Name: heuristic mining of hierarchical genotypes and accessory genome loci in bacterial populations
Uploaded: 2021-12-07T14:30:00
Description: Watch this Scientific Journal Video about Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations at JoVE.com

This work was supported by funding provided by the UNL-IANR Agricultural Research Division and the National Institute for Antimicrobial Resistance Research and Education and by the Nebraska Food for Health Center at the Food Science and Technology Department (UNL). This research could only be completed by utilizing the Holland Computing Center (HCC) at UNL, which receives support from the Nebraska Research Initiative. We are also thankful for having access, through the HCC, to resources provided by the Open Science Grid (OSG), which is supported by the National Science Foundation and the U.S. Department of Energy&#39;s Office of Science. This work used the Pegasus Workflow Management Software which is funded by the National Science Foundation (grant #1664162).

1. Prepare input files
NOTE: The protocol is available here - https://github.com/jcgneto/jove_bacterial_population_genomics/tree/main/code.&#160;The protocol assumes that the researcher has specifically used ProkEvo (or a comparable pipeline) to get the necessary outputs available in this Figshare repository (https://figshare.com/account/projects/116625/articles/15097503 - login credentials are required - The user must create a free account to have file access!). Of note, ProkEv...

The utilization of a systems-based heuristic and hierarchical population structure analysis provides a framework to identify novel genomic signatures in bacterial datasets that have the potential to explain unique ecological and epidemiological patterns20. Additionally, the mapping of accessory genome data onto the population structure can be used to infer ancestrally-acquired and/or recently-derived traits that facilitate the spread of ST lineages or cgMLST variants across reservoirs<sup class="x...

The increasing use of bacterial whole-genome sequencing (WGS) as a basis for routine surveillance and epidemiological inquiry by Public Health laboratories and regulatory agencies has substantially enhanced pathogen outbreak investigations1,2,3,4. As a consequence, large volumes of de-identified WGS data are now publicly available and can be used to study aspects of the population biology of pathogenic species at an unprecedented scale, including studies based on: population structures, genotype frequencies, and gene/allele frequencies across multiple reservoirs, geographical regions, and types of environments5. The most commonly used WGS-guided epidemiological inquiries are based on analyses using only the shared core-genomic content, where the shared (conserved) content alone is used for genotypic classification (e.g., variant calling), and these variants become the basis for epidemiological analysis and tracing1,2,6,7. Typically, bacterial core-genome-based genotyping is carried out with multi-locus sequence typing (MLST) approaches using seven to a few thousand loci8,9,10. These MLST-based strategies encompass mapping of pre-assembled or assembled genomic sequences onto highly curated databases, thereby combining allelic information into reproducible genotypic units for epidemiological and ecological analysis11,12. For instance, this MLST-based classification can generate genotypic information at two levels of resolution: lower-level sequence types (STs) or ST lineages (7 loci), and higher-level core-genome MLST (cgMLST) variants (~ 300-3,000 loci)10.
MLST-based genotypic classification is computationally portable and highly reproducible between laboratories, making it widely accepted as an accurate sub-typing approach beneath the bacterial species level13,14. However, bacterial populations are structured with species-specific varying degrees of clonality (i.e., genotypic homogeneity), complex patterns of hierarchical kinship between genotypes15,16,17, and a wide range of variation in the distribution of accessory genomic content18,19. Thus, a more holistic approach goes beyond discrete classifications into MLST genotypes and incorporates the hierarchical relationships of genotypes at different scales of resolution, along with mapping of accessory genomic content onto genotypic classifications, which facilitates population-based inference18,20,21. Moreover, analyses can also focus on shared patterns of inheritance of accessory genomic loci among even distantly-related genotypes21,22. Overall, the combined approach enables agnostic interrogation of relationships between population structure and the distribution of specific genomic compositions (e.g., loci) among geospatial or environmental gradients. Such an approach can yield both fundamental and practical information about the ecological characteristics of specific populations that may, in turn, explain their tropism and dispersion patterns across reservoirs, such as food animals or humans.
This systems-based hierarchical population-oriented approach demands large volumes of WGS data for sufficient statistical power to predict distinguishable genomic signatures. Consequently, the approach requires a computational platform capable of processing many thousands of bacterial genomes at once. Recently, ProkEvo was developed and is a freely available, automated, portable, and scalable bioinformatics platform that allows for integrative hierarchical-based bacterial population analyses, including pan-genomic mapping20. ProkEvo allows for the study of moderate-to-large scale bacterial datasets while providing a framework to generate testable and inferable epidemiological and ecological hypotheses and phenotypic predictions that can be customized by the user. This work complements that pipeline in providing a guide on how to utilize ProkEvo-derived output files as input for analyses and interpretation of hierarchical population classifications and accessory genomic mining. The case study presented here utilized the population of Salmonella enterica lineage I zoonotic serovar S. Newport as an example and was specifically aimed at providing practical guidelines for microbiologists, ecologists, and epidemiologists on how to: i) use an automated phylogeny-dependent approach to map hierarchical genotypes; ii) assess the frequency distribution of genotypes as a proxy for evaluating ecological fitness; iii) determine lineage-specific degrees of clonality using independent statistical approaches; and iv) map lineage-differentiating AMR loci as an example of how to mine accessory genomic content in the context of the population structure. More broadly, this analytical approach provides a generalizable framework to perform a population-based genomic analysis at a scale that can be used to infer evolutionary and ecological patterns regardless of the targeted species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>amr_data_filtered</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28758762</td>
		</tr>
		<tr>
			<td>amr_data_raw</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28547994</td>
		</tr>
		<tr>
			<td>baps_output</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28548003</td>
		</tr>
		<tr>
			<td>Core-genome phylogeny</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28548006</td>
		</tr>
		<tr>
			<td>genome_sra</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28639209</td>
		</tr>
		<tr>
			<td>Linux, Mac, or PC</td>
			<td></td>
			<td></td>
			<td>any high-performance platform</td>
		</tr>
		<tr>
			<td>mlst_output</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28547997</td>
		</tr>
		<tr>
			<td>sistr_output</td>
			<td></td>
			<td></td>
			<td>https://figshare.com/account/projects/116625/articles/14829225?file=28548000</td>
		</tr>
		<tr>
			<td>figshare credentials are required for login and have access to the files</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

heuristic mining of hierarchical genotypes and accessory genome loci in bacterial populations

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried out by Public Health laboratories and regulatory agencies. Large volumes of publicly available WGS data can be used to study pathogenic populations at a large scale. Recently, a freely available computational platform called ProkEvo was published to enable reproducible, automated, and scalable hierarchical-based population genomic analyses using bacterial WGS data. This implementation of ProkEvo demonstrated the importance of combining standard genotypic mapping of populations with mining of accessory genomic content for ecological inference. In particular, the work highlighted here used ProkEvo-derived outputs for population-scaled hierarchical analyses using the R programming language. The main objective was to provide a practical guide for microbiologists, ecologists, and epidemiologists by showing how to: i) use a phylogeny-guided mapping of hierarchical genotypes; ii) assess frequency distributions of genotypes as a proxy for ecological fitness; iii) determine kinship relationships and genetic diversity using specific genotypic classifications; and iv) map lineage differentiating accessory loci. To enhance reproducibility and portability, R markdown files were used to demonstrate the entire analytical approach. The example dataset contained genomic data from 2,365 isolates of the zoonotic foodborne pathogen Salmonella Newport. Phylogeny-anchored mapping of hierarchical genotypes (Serovar -&#62; BAPS1 -&#62; ST -&#62; cgMLST) revealed the population genetic structure, highlighting sequence types (STs) as the keystone differentiating genotype. Across the three most dominant lineages, ST5 and ST118 shared a common ancestor more recently than with the highly clonal ST45 phylotype. ST-based differences were further highlighted by the distribution of accessory antimicrobial resistance (AMR) loci. Lastly, a phylogeny-anchored visualization was used to combine hierarchical genotypes and AMR content to reveal the kinship structure and lineage-specific genomic signatures. Combined, this analytical approach provides some guidelines for conducting heuristic bacterial population genomic analyses using pan-genomic information.

By utilizing the computational platform ProkEvo for population genomics analyses, the first step in bacterial WGS data mining is comprised of examining the hierarchical population structure in the context of a core-genome phylogeny (Figure 1). In the case of S. enterica lineage I, as exemplified by the S. Newport dataset, the population is hierarchically structured as follows: serovar (lowest level of resolution), BAPS1 sub-groups or haplotypes, ST lineages, and cg...

Watch this Scientific Journal Video about Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations at JoVE.com

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations

This analytical computational platform provides practical guidance for microbiologists, ecologists, and epidemiologists interested in bacterial population genomics. Specifically, the work presented here demonstrated how to perform: i) phylogeny-guided mapping of hierarchical genotypes; ii) frequency-based analysis of genotypes; iii) kinship and clonality analyses; iv) identification of lineage differentiating accessory loci.

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried ...

heuristic-mining-hierarchical-genotypes-accessory-genome-loci

Research

Education

JoVE Core

JoVE Journal

Statistics

Genetics

Unit 1

Biostatistics

SCIENTISTS IN ACTION

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

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 ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Screening Cotton Genotypes for Reniform Nematode Resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

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 ...