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Methods in Molecular Biology

Jaana Koistinen, Mervi Sjöblom, Kristian Spilling
Being able to measure total nitrogen (TN) is important for following the nitrogen budget. In this chapter, we present the spectrophotometric method we use for determining TN. The method relies on oxidation and reduction steps, involving persulfate digestion of nitrogen compounds into nitrate followed by spectrophotometric determination.
February 9, 2019: Methods in Molecular Biology
Renate Gehwolf, Gabriel Spitzer, Andrea Wagner, Christine Lehner, Nadja Weissenbacher, Herbert Tempfer, Andreas Traweger
Tendons harbor various cell populations, including cells displaying classical adult mesenchymal stromal cell criteria. Previous studies have shown that a tenogenic phenotype is more effectively maintained in a 3D cell culture model under mechanical load. This chapter describes a method to isolate tendon-derived cells from rat Achilles tendons and the subsequent formation of 3D-embedded cell cultures. These tendon-like constructs can then be analyzed by various means, including histology, immunohistochemistry, qPCR, or standard protein analysis techniques...
February 8, 2019: Methods in Molecular Biology
Keai Sinn Tan, Nathalie Brouard, Daisuke Sugiyama
The development, differentiation, and maturation of hematopoietic cells are regulated by the intrinsic and extrinsic regulation. Intrinsic activity is affected by cell autonomous gene expression and extrinsic factors originate from the so-called niche surrounding the hematopoietic cells. It remains unclear why the hematopoietic sites are shifted during embryogenesis. Flow cytometry and immunohistochemistry enable us to study embryonic regulation of hematopoietic niche in the mouse embryo.
January 23, 2019: Methods in Molecular Biology
Mohammad Pooya Naghshbandi, Meisam Tabatabaei, Mortaza Aghbashlo, Muhammad Nauman Aftab, Irfana Iqbal
Microalgae are considered as promising cell factories for the production of various types of biofuels, including bioethanol, biodiesel, and biohydrogen by using carbon dioxide and sunlight. In spite of unique advantages of these microorganisms, the commercialization of microalgal biofuels has been hindered by poor economic features. Metabolic engineering is among the most promising strategies put forth to overcome this challenge. In this chapter, metabolic pathways involved in lipid and hydrogen production by microalgae are reviewed and discussed...
January 22, 2019: Methods in Molecular Biology
Joydeep Ghosh, Safa F Mohamad, Edward F Srour
Hematopoietic stem cells (HSCs) are regulated by multiple components of the hematopoietic niche, including bone marrow-derived macrophages and osteomacs. However, both macrophages and osteomacs are phenotypically similar. Thus, specific phenotypic markers are required to differentially identify the effects of osteomacs and bone marrow macrophages on different physiological processes, including hematopoiesis and bone remodeling. Here, we describe a protocol for isolation of murine bone marrow-derived macrophages and osteomacs from neonatal and adult mice and subsequent identification by multi-parametric flow cytometry using an 8-color antibody panel...
December 12, 2018: Methods in Molecular Biology
Anne-Sophie Mertgen, Markus Rottmar, Lukas Weidenbacher, Anne Géraldine Guex
Endothelial cell culture under flow, to mimic physiological conditions within blood vessels, has gained particular attention for the formation of a homogeneous endothelium in vitro. Here, we report on the design of a setup for simultaneous culture of up to nine electrospun membranes or thin polymer films in custom-made holders under flow on an orbital shaker. The versatile design of the device allows for the use of electrospun membranes/polymer films of choice and subsequent analysis with commonly used methods such as immunofluorescence or scanning electron microscopy...
December 12, 2018: Methods in Molecular Biology
Matthew Hayes
The author originally had the wrong programming command and the chapter was inadvertently published with error. The same has been updated later as below.
2019: Methods in Molecular Biology
Qian Zhu
Cells in complex tissues are organized by distinct microenvironments and anatomical structures. This spatial environment of cells is thought to be important for division of labor and other specialized functions of tissues. Recently developed spatial transcriptomic technologies enable the quantification of expression of hundreds of genes while accounting for cells' spatial coordinates, providing an opportunity to study spatially organized structures. Here, we describe a computational pipeline for detecting the spatial organization of cells based on a hidden Markov random field model...
2019: Methods in Molecular Biology
Ida Lindeman, Michael J T Stubbington
In this chapter, we describe TraCeR and BraCeR, our computational tools for reconstruction of paired full-length antigen receptor sequences and clonality inference from single-cell RNA-seq (scRNA-seq) data. In brief, TraCeR reconstructs T-cell receptor (TCR) sequences from scRNA-seq data by extracting sequencing reads derived from TCRs by aligning the reads from each cell against synthetic TCR sequences. TCR-derived reads are then assembled into full-length recombined TCR sequences. BraCeR builds on the TraCeR pipeline and accounts for somatic hypermutations (SHM) and isotype switching...
2019: Methods in Molecular Biology
Shiqi Xie, Gary C Hon
Transcriptional enhancers drive cell-type-specific gene expression patterns, and thus play key roles in development and disease. Large-scale consortia have extensively cataloged >one million putative enhancers encoded in the human genome. But few enhancers have been endogenously tested for function. For almost all enhancers, it remains unknown what genes they target and how much they contribute to target gene expression. We have previously developed a method called Mosaic-seq, which enables the high-throughput interrogation of enhancer activity by performing pooled CRISPRi-based epigenetic suppression of enhancers with a single-cell transcriptomic readout...
2019: Methods in Molecular Biology
Caleb Lareau, Divy Kangeyan, Martin J Aryee
Recent technological developments have enabled the characterization of the epigenetic landscape of single cells across a range of tissues in normal and diseased states and under various biological and chemical perturbations. While analysis of these profiles resembles methods from single-cell transcriptomic studies, unique challenges are associated with bioinformatics processing of single-cell epigenetic data, including a much larger (10-1,000×) feature set and significantly greater sparsity, requiring customized solutions...
2019: Methods in Molecular Biology
Yuanhua Huang, Guido Sanguinetti
Single-cell RNA-seq (scRNA-seq) provides a comprehensive measurement of stochasticity in transcription, but the limitations of the technology have prevented its application to dissect variability in RNA processing events such as splicing. In this chapter, we review the challenges in splicing isoform quantification in scRNA-seq data and discuss BRIE (Bayesian regression for isoform estimation), a recently proposed Bayesian hierarchical model which resolves these problems by learning an informative prior distribution from sequence features...
2019: Methods in Molecular Biology
Meichen Dong, Yuchao Jiang
Allele-specific expression is traditionally studied by bulk RNA sequencing, which measures average gene expression across cells. Single-cell RNA sequencing (scRNA-seq) allows the comparison of expression distribution between the two alleles of a diploid organism, and characterization of allele-specific bursting. Here we describe SCALE, a bioinformatic and statistical framework for allele-specific gene expression analysis by scRNA-seq. SCALE estimates genome-wide bursting kinetics at the allelic level while accounting for technical bias and other complicating factors such as cell size...
2019: Methods in Molecular Biology
Alicia T Lamere, Jun Li
Single-cell RNA-Sequencing is a pioneering extension of bulk-based RNA-Sequencing technology. The "guilt-by-association" heuristic has led to the use of gene co-expression networks to identify genes that are believed to be associated with a common cellular function. Many methods that were developed for bulk-based RNA-Sequencing data can continue to be applied to single-cell data, and several of the most widely used methods are explored. Several methods for leveraging the novel time information contained in single-cell data when constructing gene co-expression networks, which allows for the incorporation of directed associations, are also discussed...
2019: Methods in Molecular Biology
Weiyan Chen, Andrew E Teschendorff
The ability to measure molecular properties (e.g., mRNA expression) at the single-cell level is revolutionizing our understanding of cellular developmental processes and how these are altered in diseases like cancer. The need for computational methods aimed at extracting biological knowledge from such single-cell data has never been greater. Here, we present a detailed protocol for estimating differentiation potency of single cells, based on our Single-Cell ENTropy (SCENT) algorithm. The estimation of differentiation potency is based on an explicit biophysical model that integrates the RNA-Seq profile of a single cell with an interaction network to approximate potency as the entropy of a diffusion process on the network...
2019: Methods in Molecular Biology
Zhicheng Ji, Hongkai Ji
In many single-cell RNA-seq (scRNA-seq) experiments, cells represent progressively changing states along a continuous biological process. A useful approach to analyzing data from such experiments is to computationally order cells based on their gradual transition of gene expression. The ordered cells can be viewed as samples drawn from a pseudo-temporal trajectory. Analyzing gene expression dynamics along the pseudotime provides a valuable tool for reconstructing the underlying biological process and generating biological insights...
2019: Methods in Molecular Biology
Jean Fan
Integrating prior knowledge of pathway-level information can enhance power and facilitate interpretation of gene expression data analyses. Here, we provide a practical demonstration of the value of gene set or pathway enrichment testing and extend such techniques to identify and characterize transcriptional subpopulations from single-cell RNA-sequencing data using pathway and gene set overdispersion analysis (PAGODA).
2019: Methods in Molecular Biology
Huiyu Sun, Yincong Zhou, Lijiang Fei, Haide Chen, Guoji Guo
For decades, people have been trying to define cell type with the combination of expressed genes. The choice of the limited number of genes for the classification limits the precision of this system. Here, we build a "single-cell Mouse Cell Atlas (scMCA) analysis" pipeline based on scRNA-seq datasets covering all mouse cell types. We build the scMCA reference and then use the tool "scMCA" to match single-cell digital expression to its closest cell type.
2019: Methods in Molecular Biology
Lan Jiang
High-throughput single-cell technologies have great potential to discover new cell types. Here, we present a novel computational method, called GiniClust (Jiang et al., Genome Biol 17(1):144, 2016), to overcome the challenge of detecting rare cell types that are distinct from a large population.
2019: Methods in Molecular Biology
Karthik Shekhar, Vilas Menon
Unprecedented technological advances in single-cell RNA-sequencing (scRNA-seq) technology have now made it possible to profile genome-wide expression in single cells at low cost and high throughput. There is substantial ongoing effort to use scRNA-seq measurements to identify the "cell types" that form components of a complex tissue, akin to taxonomizing species in ecology. Cell type classification from scRNA-seq data involves the application of computational tools rooted in dimensionality reduction and clustering, and statistical analysis to identify molecular signatures that are unique to each type...
2019: Methods in Molecular Biology
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