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

Summary

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.

Abstract

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.

Introduction

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 ecology^1,2,3. 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 ....

Protocol

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

1. Moisten a cotton ball with an isoflurane anesthetic or another available anesthetic.
2. 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 comfortably.
3. After several minutes of allowing the bag to permeate with anesthetic, place a bat bag containing the bat into the plastic bag....

Results

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 (<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 °C. Extractions were carried out us.......

Discussion

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.......

Materials

List of materials used in this article

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