Reviewer
Ning Liu
  • Faculty, Institute of Microbiology, Chinese Academy of Sciences
Research fields
  • Plant Science
Simplified Epigenome Profiling Using Antibody-tethered Tagmentation
Authors:  Steven Henikoff, Jorja G. Henikoff and Kami Ahmad, date: 06/05/2021, view: 5210, Q&A: 0

We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNA in situ. We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.

mRNA Extraction from Gill Tissue for RNA-sequencing
Authors:  Jukka-Pekka Verta and Felicity Jones, date: 03/05/2020, view: 4517, Q&A: 0
Adaptation is thought to proceed in part through spatial and temporal changes in gene expression. Fish species such as the threespine stickleback are powerful vertebrate models to study the genetic architecture of adaptive changes in gene expression since divergent adaptation to different environments is common, they are abundant and easy to study in the wild and lab, and have well-established genetic and genomic resources. Fish gills, due to their respiratory and osmoregulatory roles, show many physiological adaptations to local water chemistry, including differences in gene expression. However, obtaining high-quality RNA using popular column-based extraction methods can be challenging from small tissue samples high in cartilage and bone such as fish gills. Here, we describe a bead-based mRNA extraction and transcriptome RNA-seq protocol that does not use purification columns. The protocol can be readily scaled according to sample size for the purposes of diverse gene expression experiments using animal or plant tissue.
Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen
We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched fraction was then subjected to shotgun proteomics for identification of proteins, followed by in silico analysis to annotate and classify the detected proteins.
Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis
Authors:  Liangsheng Wang and Dong Liu, date: 04/05/2017, view: 11324, Q&A: 0
Induction and secretion of acid phosphatase (APase) is a universal adaptive response of higher plants to low-phosphate stress (Tran et al., 2010). The intracellular APases are likely involved in the remobilization and recycling of phosphate (Pi) from intracellular Pi reserves, whereas the extracellular or secreted APases are believed to release Pi from organophosphate compounds in the rhizosphere. The phosphate starvation-induced secreted APases can be released into the rhizosphere or retained on root surfaces (root-associated APases). In this article, we describe the protocols for analyzing root-secreted APase activity in the model plant Arabidopsis thaliana (Arabidopsis). In Arabidopsis, the activity of both root-associated APases and APases that are released into the rhizosphere can be quantified based on their ability to cleave a synthesized substrate, para-nitrophenyl-phosphate (pNPP), which releases a yellow product, para-nitrophenol (pNP) (Wang et al., 2011 and 2104). The root-associated APase activity can also be directly visualized by applying a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), to the root surface (Lloyd et al., 2001; Tomscha et al., 2004; Wang et al., 2011 and 2014) whereas the isozymes of APases that are released into rhizosphere can be profiled using an in-gel assay (Trull and Deikman, 1998; Tomscha et al., 2004; Wang et al., 2011 and 2014). The protocol for analysis of intracellular APase activity in Arabidopsis has been previously described (Vicki and William, 2013).
Documentation of Floral Secretory Glands in Pleurothallidinae (Orchidaceae) Using Scanning Electron Microscopy (SEM)
Authors:  Adam P. Karremans, Bertie Joan van Heuven, Rob Langelaan and Barbara Gravendeel, date: 11/20/2016, view: 9464, Q&A: 0
A clear, step by step description of the treatment of orchid flowers, subtribe Pleurothallidinae, with Critical Point Drying for SEM is presented. It shows that a simple, short fixation and dehydration method prior to Critical Point Drying is sufficient to obtain good results.
Measurement of Uptake and Root-to-Shoot Distribution of Sulfate in Arabidopsis Seedlings
Authors:  Naoko Yoshimoto, Tatsuhiko Kataoka, Akiko Maruyama-Nakashita and Hideki Takahashi, date: 01/05/2016, view: 8970, Q&A: 0
Sulfur is an essential macronutrient required for growth and development of plants. Plants take up sulfate from the soil environment through the function of plasma membrane-bound sulfate transporters expressed at the root surface cell layers. Plants then utilize the incorporated sulfate as the main sulfur source to synthesize sulfur-containing compounds such as cysteine and methionine. Measurement of root sulfate uptake capacity is essential for analyzing mutants showing altered levels of sulfate transporters and/or sulfur metabolic enzymes as a result of genetic modification or due to the effect of intrinsic or environmental factors modulating their gene expression. The method described in this protocol allows quantitative investigation of sulfate uptake rates and root-to-shoot sulfate distribution in Arabidopsis seedlings using [35S] sulfate as a radioactive tracer. The method is designed for parallel comparisons of multiple Arabidopsis accessions, mutants or transgenic lines at the seedling stage.
Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy
Authors:  Teagen D. Quilichini, A. Lacey Samuels and Carl J. Douglas, date: 12/05/2015, view: 9533, Q&A: 0
The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. The technique described was optimized for the detection of autofluorescent components of the pollen wall, including sporopollenin and the pollen coat, and provided spatial and developmental data on the autofluorescent metabolites in anthers of wild-type and pollen wall mutant plants (Quilichini et al., 2014). The use of two-photon imaging of live, intact anthers holds potential for future studies aimed at understanding the spatial relationship between gametophytic and sporophytic tissues during pollen development and the distribution of metabolites or fluorescently-tagged proteins within developing anthers.
Detection of Poly (A) RNA in Mesophyll Cells of Nicotiana benthamiana Using in situ Hybridization
Authors:  Yuri Mizuno and Daigo Takemoto, date: 09/05/2015, view: 8550, Q&A: 0
Export of transcribed mRNAs from nucleoplasm to cytosol is an essential process for the translation of genes into proteins. This process is tightly regulated by nuclear pores, composed of about 30 nucleoporin proteins (Nups). Whether or not the mRNAs are able to be appropriately exported to cytoplasm is of an importance for understanding the role of Nups. Here, we describe a practical protocol to detect the intracellular localization of mRNAs in mesophyll cells of Nicotiana benthamiana (N. benthamiana). This protocol is based on poly (A) in situ hybridization method using an oligo d(T) probe conjugated with Alexa Fluor-488.
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