Current protocols最新文献

Globally, porcine rotavirus is a leading cause of gastroenteritis in nursing and post-weaning piglets, as well as adult pigs. Between February 2015 and June 2016, 156 fecal samples were collected from pigs in the Northeastern part of Accra, Ghana, and screened for Group A rotavirus using the Proflow^TM Kit. Here, we describe different extraction methods that were employed to recover high-quality RNA for downstream analysis, with emphasis on a novel hybrid extraction method. The hybrid approach with a kit and manual extraction method led to a 10-fold greater RNA yield versus the kit-based method alone. The new extraction method gave an average purity ratio (A₂₆₀/A₂₈₀) of 1.8, which was also significantly higher than that obtained solely from the manual or kit-based extraction methods. Our novel hybrid approach will be useful in the extraction of rotavirus from animal fecal samples, thus improving the yield of RNA for downstream analysis. © 2024 Wiley Periodicals LLC. Basic Protocol: Hybrid 2: A double lysis method for RNA extraction from animal stool samples Support Protocol 1: The GenElute extraction method Support Protocol 2: Hybrid 1 extraction method.

Single-cell genomic analysis enables researchers to gain novel insights across diverse research areas, including developmental biology, tumor heterogeneity, and disease pathogenesis. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging as the amount of genomic DNA present in a single cell is limited. Advancements in multiple displacement amplification (MDA) technologies allow the unbiased amplification of limited quantities of DNA under conditions that maintain its integrity. This method of amplification results in high yield and facilitates the generation of high-complexity NGS libraries that ensure the highest coverage to effectively allow variant calling.

With the introduction of new sequencing platforms and chemistry, whole genome sequencing became a more cost-effective application, but enrichment of specific regions of interest further reduces the amount of required sequencing output and associated costs. There are two enrichment methods, polymerase chain reaction (PCR)–based and hybrid-capture-based methods. PCR-based methods are very flexible and highly effective but focus on specific loci, typically known to be associated with disease. Inherited diseases of unknown genetic origin require a more comprehensive approach to capture the genetic variation that is not yet associated with a specific disease. Hybrid capture enrichment methods require considerable amounts of DNA such that exome enrichment from single cells is only possible after preamplification of this limited material.

This article describes the complete workflow from single cells and small quantities of DNA to exome-NGS libraries for Illumina sequencing instruments and includes the following protocols: © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Whole genome amplification from single cells or small amounts of gDNA

Basic Protocol 2: NGS library generation of MDA-amplified material

Basic Protocol 3: Exome enrichment

This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes. Proteins can be purified directly from clarified lysate, non-clarified lysate, and even non-lysed cultures without concerns of system clogging. Strong binding between the Ni²⁺ and the target protein's His₆-tag effectively captures the target protein on IMAC resins or membranes placed in the VFD. Continuous flow of different solutions through the VFD allows dynamic binding, washing, and elution of the target protein. Furthermore, the system dramatically accelerates protein purification; a typical purification from cell lysate requires approximately 4 min. Herein, we demonstrate the single-step purification of two His₆-tagged proteins from both clarified and non-clarified cell lysates without requiring batch binding. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation of the resin-loaded membrane insert and the vortex fluidic device (VFD) setup prior to purification

Basic Protocol 2: Purification of His₆-tagged proteins using the VFD

Alternate Protocol: VFD-mediated His₆-tagged protein purification from non-clarified lysate

Support Protocol: Preparation of chemically modified glass fiber membrane for VFD-mediated immobilized metal affinity chromatography purification

LipidOne 2.0 (https://lipidone.eu) is a new web bioinformatic tool for the analysis of lipidomic data. It facilitates the exploration of the three structural levels of lipids: classes, molecular species, and lipid building blocks (acyl, alkyl, or alkenes chains). The tool's flexibility empowers users to seamlessly include or exclude experimental groups and lipid classes at any stage of the analysis. LipidOne 2.0 offers a range of mono- and multivariate statistical analyses, specifically tailored to each structural level. This includes a novel lipid biomarker identification function, integrating four diverse statistical parameters. LipidOne 2.0 incorporates Lipid Pathway analysis across all three structural levels of lipids. Users can identify lipid-involved reactions through case-control comparisons, generating lists of genes/enzymes and their activation states based on Z scores. Accessible without the need for registration, LipidOne 2.0 provides a user-friendly and efficient platform for exploring and analyzing lipidomic data. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Dataset preparation for LipidOne 2.0

Support Protocol: Lipid nomenclature from spectrometric experiments

Basic Protocol 2: Uploading a dataset into LipidOne 2.0

Basic Protocol 3: Data mining of lipidomic dataset by LipidOne 2.0

Primary human hepatocytes (PHHs) are recognized as the "gold standard" for evaluating toxicity of various drugs or chemicals in vitro. However, due to their limited availability, primary hepatocytes isolated from rodents are more commonly used in various experimental studies than PHHs. However, bigger differences in drug metabolism were seen between humans and rats compared to those between human and non-human primates. Here, we describe a method to isolate primary hepatocytes from the liver of rhesus macaques (Macaca mulatta, a species of Old-World monkey) after in situ whole liver perfusion. Techniques for cryopreserving and recovering primary macaque hepatocytes (PMHs) are also described. Given the remarkable physiological and genetic similarity of non-human primates to humans, PMHs isolated using this protocol may serve as a reliable surrogate of PHHs in toxicological research and preclinical studies. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

Basic Protocol 1: In situ whole liver perfusion

Basic Protocol 2: Primary macaque hepatocyte isolation and cell plating

Basic Protocol 3: Cryopreservation and recovery of primary macaque hepatocytes

The study of human intestinal physiology and host-microbe interactions is crucial for understanding gastrointestinal health and disease. Traditional two-dimensional cell culture models lack the complexity of the native intestinal environment, limiting their utility in studying intestinal biology. Here, we present a detailed protocol for the set up and utilization of a three-dimensional (3D) in vitro bioreactor system for human intestinal studies and bacterial co-culture. This article outlines the design and assembly of the bioreactor system, scaffold fabrication, bacterial culture techniques, analysis methods, and troubleshooting tips. By providing step-by-step instructions, the goal is to enable other laboratories to utilize physiologically relevant tissue models of the human intestine, incorporating key features, such as nutrient flow, multiple human cell types, 3D architecture, and microbial communities. The incorporation of commensal bacteria into the bioreactor system allows for the investigation of complex host-microbe interactions, providing insight into gastrointestinal health and pathology. This article serves as a comprehensive resource for scientists seeking to advance their understanding of intestinal biology toward the development of novel therapeutic strategies for gastrointestinal disorders. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Scaffold design

Basic Protocol 2: Intestinal cell culture: Caco2 cells

Basic Protocol 3: Intestinal cell culture: organoids

Basic Protocol 4: Bioreactor design and set up

Basic Protocol 5: Bacteria in 3D bioreactor set up

Basic Protocol 6: Bacteria and drug dosing

Protoplast sorting and purification methods are powerful tools enabling the enrichment of cellular subpopulations for basic and applied studies in plant sciences. Fluorescence-activated protoplast sorting (FAPS) is an efficient method to isolate specific protoplast populations based on innate features (size and autofluorescence) or expression of fluorescent proteins. FAPS-based methods have recently been deployed in single-cell purification for single-cell RNA sequencing–based transcriptional profiling studies. Protoplast sorting methods integrated with the ability to culture and recover whole plants add value to functional genomics and gene editing applications. Enriching cells expressing nucleases linked to fluorescent proteins can maximize knockout or knockin editing efficiencies and minimize toxic and off-target effects. Here, we report the protocol for protoplast preparation, sterile cell sorting, culture, and downstream regeneration of plants from canola protoplasts. This protocol can be successfully applied to all totipotent protoplast methods that can regenerate into whole plants. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of transfected canola protoplasts for sorting

Basic Protocol 2: Fluorescence-activated protoplast sorting

Basic Protocol 3: Bead culture of sorted protoplasts and recovery of plantlets

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples

Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system

Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery

DNA methylation is well-established as a major epigenetic mechanism that can control gene expression and is involved in both normal development and disease. Analysis of high-throughput-sequencing-based DNA methylation data is a step toward understanding the relationship between disease and phenotype. Analysis of CpG methylation at single-base resolution is routinely done by bisulfite sequencing, in which methylated Cs remain as C while unmethylated Cs are converted to U, subsequently seen as T nucleotides. Sequence reads are aligned to the reference genome using mapping tools that accept the C-T ambiguity. Then, various statistical packages are used to identify differences in methylation between (groups of) samples. We have previously developed the Differential Methylation Analysis Pipeline (DMAP) as an efficient, fast, and flexible tool for this work, both for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS). The protocol described here includes a series of scripts that simplify the use of DMAP tools and that can accommodate the wider range of input formats now in use to perform analysis of whole-genome-scale DNA methylation sequencing data in various biological and clinical contexts. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: DMAP2 workflow for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS)

The lung comprises multiple components including the parenchyma, airways, and visceral pleura, where each constituent displays specific material properties that together govern the whole organ's properties. The structural and mechanical complexity of the lung has historically undermined its comprehensive characterization, especially compared to other biological organs, such as the heart or bones. This knowledge void is particularly remarkable when considering that pulmonary disease is one of the leading causes of morbidity and mortality across the globe. Establishing the mechanical properties of the lung is central to formulating a baseline understanding of its operation, which can facilitate investigations of diseased states and how the lung will potentially respond to clinical interventions. Here, we present established and widely accepted experimental protocols for pulmonary material quantification, specifying how to extract, prepare, and test each type of lung constituent under planar biaxial tensile loading to investigate the mechanical properties, such as physiological stress–strain profiles, anisotropy, and viscoelasticity. These methods are presented across an array of commonly studied species (murine, rat, and porcine). Additionally, we highlight how such material properties may inform the construction of an inverse finite element model, which is central to implementing predictive computational tools for accurate disease diagnostics and optimized medical treatments. These presented methodologies are aimed at supporting research advancements in the field of pulmonary biomechanics and to help inaugurate future novel studies. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: General procedures in lung biaxial testing

Alternate Protocol 1: Parenchymal-specific preparation and loading procedures

Alternate Protocol 2: Airway-specific preparation and loading procedures

Alternate Protocol 3: Visceral pleura–specific preparation and loading procedures

Basic Protocol 2: Computational analysis