STAR Protocols最新文献

Here, we present a protocol for evaluating glucose metabolism in mouse retinas and retinal pigment epithelium (RPE)-choroid tissue by tracking the incorporation of ¹³C₆ from U-¹³C₆-glucose with gas chromatography-mass spectrometry (GC-MS). We describe steps for incubating tissues in Krebs-Ringer bicarbonate solution and homogenizing tissues. We then detail procedures for extracting metabolites and determining isotopic labeling of intermediates in glycolysis and the tricarboxylic acid (TCA) cycle using GC-MS. The approach has been adapted to study glucose metabolism in various tissues, animal models, and genetic conditions. For complete details on the use and execution of this protocol, please refer to Nolan et al.¹.

RNA-protein interactions drive gene regulation, subcellular organization, and noncoding RNA function. Here, we present a protocol for measuring RNA-protein associations in formaldehyde-crosslinked mammalian cells using RNA immunoprecipitation followed by sequencing (RIP-seq) and quantitative PCR (RIP-qPCR). We include steps and best practices for qualifying reagents, preparing cells, and processing and analyzing data, including a standardized set of steps to quantify signal over noise. This protocol is broadly applicable for the study of RNA-protein interactions in cells. For complete details on the use and execution of this protocol, please refer to Trotman et al.¹.

Spatial analysis of cells and their microenvironment within tissues enhances our understanding of biological processes. Ideally, a broad range of biomolecules should be analyzed in large 3D tissue specimens at subcellular resolution. Here, we present a protocol to identify and extract target sections from previously cleared tissues. We describe steps for combining 3D light sheet imaging and subsequent 3D-guided deep cell phenotyping via multi-cyclic 2D microscopy.

Bacterial coaggregation is the specific recognition and adhesion of genetically distinct bacteria, and it is central to multispecies biofilm formation. Here, we present a protocol integrating visual, turbidimetry, epifluorescence microscopy, and thermodynamic analysis to study coaggregation. We describe procedures for performing coaggregation assays and analyzing complementary data from different assays. This integrated workflow provides a robust and multidimensional framework for investigating bacterial coaggregation in different contexts. For complete details on the use and execution of this protocol, please refer to Afonso et al.^1,2.

Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis. For complete details on the use and execution of this protocol, please refer to Michael et al.¹.

The large-scale production of functional hepatocytes is critical for liver disease treatment, disease modeling, and drug development. Here, we present a protocol for isolating, expanding, and maintaining human gallbladder epithelial cells (hGBECs). We describe steps for characterizing hGBECs and their differentiation into hepatocytes. We then detail procedures to assess the functionality of the derived hepatocytes using key hepatic function assays. For complete details on the use and execution of this protocol, please refer to Chen et al.¹.

Vascular organoids (VOs) are 3D multicellular constructs that model vascular development and function. Here, we present a protocol to generate VOs from human induced pluripotent stem cells via orthogonal activation of transcription factors. We describe steps for inducing endothelial and mural lineages independently using doxycycline-inducible expression of ETV2 and NKX3.1. This protocol enables efficient formation of functional VOs within 5 days, streamlining conventional techniques and supporting applications in vascular biology, tissue engineering, and regenerative medicine. For complete details on the use and execution of this protocol, please refer to Gong et al.¹.

Mesenchymal stem/stromal cells (MSCs) are known for their regenerative properties. This protocol describes a co-culture system for investigating molecular interactions between MSCs and intestinal epithelial organoids following injury. We outline steps for assessing the immediate effects of MSCs on organoid growth and survival, as well as a model for evaluating longer term responses. The workflow is adaptable and can be readily modified to examine MSC interactions with additional cell types or in different injury contexts. For complete information on the use and execution of this protocol, please refer to Yetkin-Arik et al.

Mechanical forces influence a range of cellular behaviors; however, how these forces are sensed and converted into biochemical changes remains incompletely understood. A key aspect of mechanotransduction is the regulation of subcellular protein localization. Here, we present a protocol describing the engineering of cell lines with tunable actomyosin contractility combined with a proximity biotinylation strategy confined to the nucleus followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This approach allows the identification of proteins whose nuclear localization is controlled by changes of actomyosin contractility. For complete details on the use and execution of this protocol, please refer to Tseng et al.¹.

Here, we present a reproducible protocol for estimating cross-ancestry local genetic correlation using Logica, a likelihood-based framework that employs summary statistics from genome-wide association studies (GWASs) and ancestry-specific linkage disequilibrium (LD). We describe steps for estimating locus-level heritability and cross-ancestry genetic correlation and outlining required inputs. We then detail analytical procedures to enable accurate and scalable inference of shared genetic architecture. For complete details on the use and execution of this protocol, please refer to Gao et al.¹.