Lipidomics has been defined as the large-scale analysis of lipids in organelles, cells, tissues, or whole organisms. Including the temporal aspects of lipid metabolic changes into this analysis allows to access yet another important aspect of lipid regulation. The resulting methodology, circadian lipidomics, has thus emerged as a novel tool to address the enormous complexity, which is present among cellular lipids. Here, we describe how mass spectrometry-based circadian lipidomics can be applied to study the impact of peripheral clocks on lipid metabolism in human primary cells and tissues, exemplified by studies in human pancreatic islets and skeletal myotubes.Lipidomics approaches provide quantitative characterization of hundreds of lipid species from biological samples. Recent studies highlight the value of these methods in studying circadian biology, and their potential goes far beyond studying lipid metabolism per se. For example, lipidomics analyses of subcellular compartments can be used to determine daily rhythmicity of different organelles and their intracellular dynamics. In this chapter we describe in detail the procedure for around the clock shotgun lipidomics, from sample preparation to bioinformatics analyses. Sample preparation includes biochemical fractionation of nuclei and mitochondria from mouse liver harvested throughout the day. Lipid content is determined and quantified, in unbiased manner and with wide coverage, using multidimensional mass spectrometry shotgun lipidomics (MDMS-SL). Circadian parameters are then determined with nonparametric statistical tests. Overall, the approach described herein is applicable for various animal models, tissues, and organelles, and is expected to yield new insight on various aspects of circadian biology and lipid metabolism.Metabolites like melatonin are essential in determining circadian phase. In the recent years, comprehensive metabolome analyses have unveiled entire panels of small biomolecules fluctuating in a circadian fashion, thus enabling a more precise determination of inner time and understanding of how circadian clock operates at the molecular level. Emerging analytical techniques allowing for the determination of exhaled metabolites in breath show promise to gain further insights noninvasively and in vivo into circadian metabolism.Circadian gene transcription transmits timing information and drives cyclic physiological processes across various tissues. Recent studies indicate that oscillating enhancer activity is a major driving force of rhythmic gene transcription. Functional circadian enhancers can be identified in an unbiased manner by correlation with the rhythms of nearby gene transcription.Global run-on sequencing (GRO-seq) measures nascent transcription of both pre-mRNAs and enhancer RNAs (eRNAs) at a genome-wide level, making it a unique tool for unraveling complex gene regulation mechanisms in vivo. Here, we describe a comprehensive protocol, ranging from wet lab to in silico analysis, for detecting and quantifying circadian transcription of genes and eRNAs. Moreover, using gene-eRNA correlation, we detail the steps necessary to identify functional enhancers and transcription factors (TFs) that control circadian gene expression in vivo. While we use mouse liver as an example, this protocol is applicable for multiple tissues.Understanding the binding of regulatory proteins to their cognate genomic sites is an important step in deciphering transcriptional networks such as the circadian oscillator. Chromatin immunoprecipitation (ChIP) enables the detection and temporal analysis of such binding events in vivo. Here, we describe the individual steps from the generation of formaldehyde-cross-linked chromatin from mouse liver nuclei, fragmentation thereof, immunoprecipitation, reversal of cross-links, fragment cleanup, and detection of binding sites by real-time PCR. Depending on the quality of the employed antibody, a clear enrichment signal over the background is expected with a resolution of about 500-800 base pairs around the selected primer-probe pair.RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.Circadian rhythms are constituted by a complex dynamical system with intertwined feedback loops, molecular switches, and self-sustained oscillations. Mathematical modeling supports understanding available heterogeneous kinetic data, highlights basic mechanisms, and can guide experimental research. Here, we introduce the basic steps from a biological question to simple models providing insight into gene-regulatory mechanisms. We illustrate the general approach by three examples modeling decay processes, clock-controlled genes, and self-sustained oscillations.With the emergence of big data science, the question how we can easily collect meaningful information about circadian clock phenotypes in large human cohorts imposes itself.  https://www.selleckchem.com/products/AZD2281(Olaparib).html  Here, we describe potentials and limitations of using questionnaires, specifically the Munich ChronoType Questionnaire (MCTQ), to characterize such circadian phenotypes. We also discuss scenarios when alternative methods might be more appropriate.The forced swim and tail suspension tests are commonly used to determine the effects of circadian-related pharmacological, genetic, and environmental manipulations on depression-like behavior in rodents. Both tests involve scoring immobility of rodents in an inescapable condition. Here we describe how to set up and carry out these tests.