Name: tissue collection of bats for -omics analyses and primary cell culture
Uploaded: 2019-10-23T13:00:00
Description: Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

We thank Centro de Ecolog&#237;a y Biodiversidad CEBIO, Erika Paliza, Miluska S&#225;nchez, Jorge Carrera, Edgar Rengifo V&#225;squez, Harold Porocarrero Zarria, Jorge Ru&#237;z Leveau, Jaime Pecheco Castillo, Carlos Tello, Fanny Cornejo, and Fanny Fern&#225;ndez Melo for making tissue collection in Peru possible. In Colombia, we thank Colciencias and Instituto de Investigaci&#243;n de Recursos Biol&#243;gicos Alexander von Humboldt, as well as Alexandra Buitrago, Mailyn Gonz&#225;lez, Andr&#233;s Juli&#225;n Lozano Florez, Darwin M. Morales Martinez, Ana Maria Ospina, Adrian Pinzon, Paola Pulido-Santacruz, and Danny Rojas for making logistics, travel, capture, and filming possible. We also thank all members of Grupo Jaragua and Yolanda Leo&#769;n for making tissue collection in the Dominican Republic possible, as well as to Bernal Rodriguez Hernandez, Bernal Matarrita, and everyone at La Selva Biological Research Station in Costa Rica, for making sampling possible, and Kasia Sawicka for helping with procedures and reagents before the trip. We thank Ella Lattenkamp &#38; Lutz Wiegrebe for access and sample collection of wing punches from Phyllostomus discolor bats for cell culture generation. Phyllostomus discolor bats originated from a breeding colony in the Department Biology II of the Ludwig-Maximilians-University in Munich. Approval to keep and breed the bats was issued by the Munich district veterinary office. LMD, SJR, KTJD, and LRY were funded by NSF-DEB 1442142. LMD was funded by NSF-DEB 1838273. LRY was funded by the NSF-PRFB 1812035. SCV and PD were funded by a Max Planck Research Group Award, and a Human Frontiers Science Program (HFSP) Research grant (RGP0058/2016). SJR, JHTP, and KTJD were funded by the European Research Council (ERC Starting grant 310482 [EVOGENO]) awarded to SJR, ECT was funded by European Research Council grant (ERC-2012-StG311000).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stony Brook University (protocols # 2013-2034-NF-4.15.16-BAT, 2014-2090-NF-1.20.17-Bat, 2014-2119-NF-Bat - 6.16.17, and 2018-NF-11.12.19-Bat).
1. Euthanasia
<ol>
	<li>Moisten a cotton ball with an isoflurane anesthetic or another available anesthetic.</li>
	<li>Place the cotton ball into a resealable, airtight plastic bag. The bag should be large enough to hold a bag in a cloth bat bag...

The protocol discussed in this manuscript describe the best sampling practices for various high-throughput molecular analyses of bats. All successful -omics studies require high quality tissue, but sampling bat tissue, as well as other non-model organisms, often occurs in field conditions that cannot be set to the same standards as those of a controlled laboratory setting. Sampling often occurs in remote locations, with minimal resources, including limited access to electricity and freezers. It is difficult, and often im...

High-throughput sequencing (HTS) methods have rapidly advanced in efficiency and decreased in cost, and it is now possible to scale these approaches to hundreds or thousands of samples. Insights obtained by the application of these technologies have enormous impacts across multiple scientific disciplines, from biomedicine to evolution and ecology1,2,3. Yet, many HTS applications rely critically on high quality nucleic acids from a living source. This limitation is likely to become increasingly problematic with the development of third generation sequencing based on long-read molecules4. For these reasons, there is a need to focus efforts on establishing best practices for collecting fresh tissue samples from wild organisms outside of the laboratory, which maximizes the utility of material and reduces the number of individuals that need to be collected.
Bat1K is an international consortium of scientists with an ongoing initiative to sequence the genome of every species of bat to chromosome-level assembly5. Bats represent 20% of mammalian diversity and have exceptional adaptations that have implications for understanding aging, disease ecology, sensory biology, and metabolism5,6. Many bats are also threatened or endangered due to human exploitation7 or are rapidly declining due to pathogens8,9, and genome-level sequencing is of great importance for conservation of these species. Although Bat1K currently aims to sequence the genomes of all bat species, the standardization of collection of tissue samples for high-quality genomic sequencing remains a key challenge across the community of organismal biologists. In addition to genomic data, functional understanding of the diversity of bat adaptations requires tissue-specific transcriptome and protein analyses, often requiring separate collection protocols. Moreover, as with all taxonomic groups, while optimal tissue collection and preservation is essential for obtaining the highest quality data for -omics analyses, communicating best practices is often difficult because of rapidly changing technologies and multiple research teams working independently.
The need to adopt best practices for bat -omics research is especially urgent, given that many bat species are rare or threatened. Unlike other small mammals such as rodents and shrews, bats are long-lived, attributable to exceptional DNA repair mechanisms10 and slow reproduction11, with most species giving birth to just one or (in a few cases) two young per year. For these reasons, bat populations can be slow to recover from disturbance, and collecting many individuals from the wild is neither advisable nor feasible. In other words, protocols must be optimized to obtain the maximum amount of data for a single specimen, thereby reducing the need to unnecessarily replicate sampling efforts.
Here, this protocol focuses specifically on standardized methods for the collection and sampling of bat tissue for genomic and transcriptomic sequencing and protein analyses. Its top priority is to ensure that bat tissues are collected ethically and responsibly, ranging from the permitting process for the collection, exportation of tissues to the minimization of stress to the animal, and long-term storage conditions. Elaborate dissections have been developed with the aim of future-proofing the usefulness of different materials collected. This manuscript provides a step-by-step guide to collect bats in a humane way that is intended to minimize impact on populations and maximize scientific value. While the focus of this protocol is specifically for use in bats, many of the steps are relevant to other vertebrate taxa, especially mammals.
Tissue collection overview 
The procedure for tissue collection, including the temperature for storage and the choice of preserving agent, will be determined by the nature of any downstream analyses planned. However, it is strongly recommended that, when possible, tissue is collected under a range of methods to maximize its future utility even if no specific analysis is planned. In general, tissue is collected and preserved for subsequent analyses of either nucleic acids (DNA and RNA), or protein. For each of these applications, tissue can be optimally preserved by directly flash freezing in liquid nitrogen (LN2). However, immediate immersion in LN2 is not always possible in the field. As technology advances, resources such as specialized vials to store DNA and RNA at ambient temperatures are becoming more readily available. While we have not validated all such materials in this protocol, we encourage other researchers to comparatively analyze the performance of new materials relative to what we present here. We do provide methods to ideally preserve tissue for different applications in situations where LN2 cannot be accessed, e.g., when LN2 transport is not possible due to site access via small plane in the Amazon. In addition, we provide a method for collecting tissue from which live cells can be grown and propagated. Below we outline key considerations for collecting material for each of these respective purposes and an overview of collection methods is given in Table 1.
Tissue for DNA 
For all harvested tissue collected, the storage media will determine if it can be used for either standard or high molecular weight (HMW) DNA extraction. HMW is required for long-read sequencing and currently required to generate chromosome level genome assemblies, or a &#8220;platinum standard&#8221; genome. Low molecular weight (LMW) DNA can be extracted from flash frozen, AllProtect (henceforth referred to as &#8220;tissue stabilizing solution&#8221;), or even RNAlater (henceforth &#8220;RNA stabilizing solution&#8221;)-preserved samples (although flash frozen samples remain optimal). DNA isolated by standard laboratory methods (e.g., silica gel membrane spin columns, phenol-chloroform), may still yield DNA fragments of up to ~20 kilobases (kb). Therefore, provided there is sufficient yield, this form of isolated DNA may be used for single insert size library preparation, in which the insert size is often ~500 base-pairs (bp), and short sequence reads of ~100 bp are generated12. This DNA is particularly useful for &#8220;resequencing&#8221; projects or studies in which full-length chromosomal data is not required. HMW DNA (10-150 kb) is more challenging and can only be reliably obtained using tissue that has been rapidly flash frozen in LN2 following harvest and maintained at a maximum of -80 &#176;C until extraction.
Low molecular weight or fragmented DNA is often sufficient for targeted approaches, including gene amplification via PCR and short-read sequencing13. PCR-based investigations using LMW DNA that target only one or a few genes have been highly informative in understanding adaptation and the molecular evolution of bat sensory biology6,14, physiology15, phylogenetics5,16, and conservation17,18. Successful targeted sequence recapture of low molecular weight and fragmented DNA has also been demonstrated for numerous vertebrate groups, including bats19. These methods are often cost-effective and minimally invasive to the bat, as fecal samples and non-lethal tissue sampling via buccal swabs or wing biopsy punches are also common ways to obtain DNA for low molecular weight analyses20,21.
However, the quality depends heavily on the type of media in which the sample is stored22. After systematic and quantitative comparisons of buccal swabs and biopsy punches, wing biopsy punches have been shown to yield consistently higher levels of DNA and were less stressful to the bat during collection22. These comparisons also showed that the best results were obtained when the wing punch was preserved in indicator silica (i.e., a type of desiccant made of silica gel beads that changes color when moisture is observed) rather than in other popular storage media such as ethanol or DMSO22; although, other storage media including tissue stabilization solution were not examined. Wing punches can also be used to grow fibroblast cells in culture, such as in Kacprzyk et al.23 and as described below (see section 6). For these methods, the wing or uropatagium should be extended gently, and a clean biopsy punch, typically 3 mm in diameter, should be used to obtain the sample. This approach appears to cause no lasting damage, with scars healing over within weeks in most cases24.
HMW DNA (10-150 kb) is more challenging and is currently only reliably obtained using tissue that has been rapidly flash frozen in LN2 following harvest and maintained at a maximum of -80 &#176;C until extraction. HMW DNA (10-150 kb) is crucial for long-read DNA sequencing and therefore for de novo genome assembly. Indeed, while most commercial kits can be used to isolate some standard HMW DNA, the resulting molecule sizes often do not meet the requirements of third generation sequencing technologies [e.g., those launched by companies such as Pacific Biosciences (PacBio), Oxford Nanopore Technologies, and 10x Genomics, or through assembly methods offered by Bionano Genomics or Dovetail Genomics]. As such, there is a new demand for &#8220;ultra HMW&#8221; DNA (&#62;150 kb). When obtaining ultra HMW DNA from bats, fresh samples of liver, brain, or muscle are all suitable, but these must be immediately flash frozen in LN2 without any storage buffer or cryoprotectant. A full description of these steps is beyond the scope of this paper but are available elsewhere25.
Tissue for RNA 
RNA is a single-stranded molecule that is less stable than DNA. Although there are many forms of RNA, -omics analyses tend to focus on mRNA (messenger RNA) and small RNAs (e.g., microRNAs). Following transcription, the mRNA is spliced to form a mature transcript that contains no introns and represents the coding portion of genes/genomes. Coding genes account for a tiny fraction of the genome size (1%-2%), making targeting mRNA a cost-effective means of obtaining sequence data for genes. MicroRNAs are a class of RNAs that regulate the process of translation of mRNA into proteins and are thus important regulatory effectors. RNA transcripts can be sequenced individually, or more commonly for -omics analyses26,27,28,29,30, as part of a transcriptome; that is, the total of all RNA transcripts present in a given sample.
Sequencing can be performed following several methods (i.e., via short-read RNA-seq or long-read whole isoform Seq), allowing analysis of both RNA abundance and isoform usage. As the quantity and diversity of mRNA transcripts varies among cells and tissues, RNA sequencing makes it possible to study and compare gene expression and regulation across samples. Interest in sequencing small RNAs and whole isoform sequencing is growing, as these methods are becoming increasingly more biologically informative. Preparation of tissue samples to sequence different classes of RNA can be performed in the same way as presented in this manuscript, with only the subsequent extraction methods differing31,32. Finally, because transcriptomes offer a high coverage subset of the protein-coding genome, the assembled dataset may be useful in genome assembly and annotation, making collection of RNA-seq data across a range of different tissues an important component of the Bat1K initiative.
In contrast to DNA, RNA is chemically unstable and also targeted by RNase enzymes, which are ubiquitously present in tissue lysates as a defensive strategy against RNA-based viruses. For these reasons, the RNA fraction in cells and tissues begins to degrade shortly after the point of sampling and/or euthanasia. Preserving the RNA therefore requires steps to prevent its degradation. This typically involves preserving freshly collected tissue at 4 &#176;C in a stabilizing agent such as RNA stabilizing solution to inactivate the RNases naturally present in tissues, followed by freezing for longer term storage. As a preferred alternative, tissue can be flash frozen in LN2; although as noted above, transporting LN2 into the field and maintaining levels to prevent the tissue thawing can be logistically challenging.
Tissue for protein 
Protein composition and relative abundance vary among cells and tissues in a similar way to what was discussed for RNA; however, proteins are on average more stable than RNA. Protein identification using proteomics typically matches a fraction of, and not the whole, protein sequence, but it can supply information on expression across tissues and characterize pathogens present. As many protein sequences are conserved across mammals, bat samples for proteomics can easily be contaminated with conserved human proteins, requiring sterile protocols (e.g., gloves, forceps) during collection. While flash-freezing in LN2 is the best way to prevent the degradation of proteins, use of dry ice, -20 &#730;C freezers, and even ice are suitable if there are no other means. As temperatures increase, the risk of differential protein breakdown also rises. Stabilizing agents such as tissue stabilization solution are effective in preserving the protein fraction of tissues at room temperature and are suitable for short-term preservation (up to one week) when flash-freezing is not viable.
The enzymatic profile of a given tissue directly influences the preservation of protein therein. Tissues with low enzymatic activity such as muscle can preserve protein profiles even at the higher temperatures in a household freezer. By contrast, liver tissue is enzymatically reactive, and its proteins have higher probabilities of degrading during preparation. The growing number of protocols for obtaining human proteomic profiles from formalin-fixed paraffin-embedded (FFPE) samples suggests that paraformaldehyde fixing of tissues holds promise for low-cost protein preservation when freezing upon collection is not feasible33,34. Although highly dependent on preservation time and condition, proteins have been identified via immunohistochemistry from formalin-fixed, ethanol-preserved bat specimens35. This approach is not scalable to proteomic-level sampling but highlights the potential for formalin-fixed bat tissues to yield protein profiles when flash-freezing is unavailable and other stabilizing agents are too costly.
Tissue for cell culture 
Sampling tissue and flash-freezing offers a finite amount of material to be used, and once the material is used, it is no longer available. Alternatively, cell cultures provide live cells that can be immediately used or preserved for future studies. Cultures also facilitate expansion of cells to increase yield when tissue samples are small. It is particularly useful in cases where tissue collection is limited, such as experiments with rare species in which non-lethal sampling is essential and therefore has wide implications for conservation. Described is a protocol in which cell culture is possible via non-lethal sampling of wing membrane tissue, but culturing is possible with multiple tissue types36,37. The protocol provided here selects for adherent cells. The combination of source tissue and growth media used makes this protocol suitable to select and grow fibroblasts, but if desired, alternate protocols can be used to select for other cell types. In the context of the Bat1K project, it is predicted that for rare and threatened species, non-lethal sampling of wing membranes and expansion of samples via culturing is essential to generate the volume of DNA needed for the multiple technologies employed5.
Bat capture 
All people handling bats should be trained by a bat-competent researcher and vaccinated against rabies with a series of pre-exposure injections. If bitten, a further series of post-exposure injections is still necessary. Standard methods for capturing bats include mist nets (Figure 1) and harp traps (Figure 2). Mist nets are most commonly used and ideal for areas with low to moderate activity, as they require the most care for minimizing bat distress. Small bats are particularly vulnerable and can die from stress if not tended to quickly. Frequent net inspections minimize bat injury and mortality as well as damage to the mist net. This detail is important because proper tissue collection requires the tissue to be fresh, and improper attention to the mist nets in bats can lead to unnecessary mortality or premature mortality before the researcher can properly process samples. Because several bats can rest in a harp trap with minimal distress, this approach is ideal for areas with high bat activity, such as near a cave or large roost. Detailed instructions for proper bat capture and data processing for collection of morphological and demographic information are available in the supplemental methods.

<table>
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf Safelock tubes</td>
			<td>Fischer Scientific</td>
			<td>10509691</td>
			<td>1.5 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>62,554,002</td>
			<td>15 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>2 mL cryovials</td>
			<td>Thomas Scientific</td>
			<td>1154P75</td>
			<td>cryogenic vials for tissues to be stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>3-mm biopsy punch</td>
			<td>Medline</td>
			<td>MIL3332</td>
			<td>wing biopsy punch for cell culture</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Greiner</td>
			<td>83,392</td>
			<td>Culture vessle</td>
		</tr>
		<tr>
			<td>Allprotect Tissue Reagent</td>
			<td>Qiagen</td>
			<td>76405</td>
			<td>for fecal samples; tissue stabilizing solution</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC10/TC20 cell counter, dual chamber</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
			<td>Chambers for the count cells using the automated cell counter TC20 by Bio-Rad</td>
		</tr>
		<tr>
			<td>collagenase IV</td>
			<td>Stemcell Technologies</td>
			<td>7909</td>
			<td>For dissociation of primary cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
			<td>Prevents crystalization of water during freezing of the cells.</td>
		</tr>
		<tr>
			<td>DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher</td>
			<td>12491-015</td>
			<td>culture media</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td>Balanced salt solution used for washing cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Fischer Scientific</td>
			<td>10270106</td>
			<td>Serum-supplement for in vitro cell culture of eukaryotic cells</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate salt</td>
			<td>Sigma Aldrich</td>
			<td>G1264-250MG</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0050</td>
			<td>Freezing container which provides 1&#176;C/min cooling rate</td>
		</tr>
		<tr>
			<td>PARAFILM</td>
			<td>Sigma</td>
			<td>P7793</td>
			<td>Wrapping tubes etc for sealing</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (pen-strep), 100x</td>
			<td>Invitrogen</td>
			<td>15140130</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) 10x, pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td>salt buffer used for washes and storage of bone tissue; dilute to 10X using de-ionized water</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermo Fisher</td>
			<td>AM7021</td>
			<td>RNA stabilizing solution</td>
		</tr>
		<tr>
			<td>RNAse away</td>
			<td>Genetech</td>
			<td>83931-250mL</td>
			<td>breaks down enzymes that lead to RNA degradation</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Fisher Scientific</td>
			<td>7631-86-9</td>
			<td>dessicant agent</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td>Cell stain used to assess cell viability</td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4049</td>
			<td>For dissociation of cells during splitting</td>
		</tr>
	</tbody>
</table>

tissue collection of bats for -omics analyses and primary cell culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

DNA 
For standard low molecular weight (LMW) analyses, DNA was extracted from three neotropical bat species. Tissue samples were collected in the field from the Dominican Republic and Costa Rica, following the protocols described in this paper. Following dissection, small pieces (&#60;0.5 cm at the thinnest section) of mixed tissues (brain, liver, and intestine) were placed in RNA stabilizing solution, flash frozen, then stored at -80 &#176;C. Extractions were carrie...

Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

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