STAR Protocols最新文献

Circulating cell-free DNA (cfDNA) fragment end motif profiles are a promising biomarker in precision oncology. Here, we present a protocol for analyzing plasma cfDNA fragment end motifs from ultra-low-pass whole-genome sequencing (WGS) data. We detail a pipeline composed of sequential bash scripts for processing post-alignment BAM files. Subsequently, we outline the procedure for downstream analysis and visualization of 4-mer as well as other n-mer cfDNA end motifs in R. For complete details on the use and execution of this protocol, please refer to Liu et al.¹.

Surface availability of the dopamine (DA) transporter (DAT) critically influences DA transmission. Here, we present a protocol that describes the preparation of mouse ventral midbrain neurons, the expression of a new optical sensor, DAT-pHluorin, and the utilization of this sensor to analyze the surface availability of DAT in live neurons via fluorescent microscopy. This approach allows quantitative measures of basal surface DAT fraction under genetic backgrounds of interest and live trafficking of DAT in response to psychoactive substances. For complete details on the use and execution of this protocol, please refer to Saenz et al.¹.

Dendritic cells (DCs) play a central role in the initiation of the adaptive immune response. Here, we present a protocol for isolating and transcriptionally profiling antigen-presenting cells (APCs) from the mouse lung and mediastinal lymph nodes (MLNs) following intranasal immunization. We describe steps for preparing single-cell suspensions from the lung and MLN, along with the detection and RNA sequencing (RNA-seq) of antigen-presenting DCs. This protocol offers a broadly applicable approach for identifying variations in DC subpopulations under diverse experimental conditions. For complete details on the use and execution of this protocol, please refer to Youhui et al.¹.

This protocol describes the preparation of a nanoparticle-encapsulated bioink capable of protecting tissue-engineered constructs against bacterial infections while also providing contrast for magnetic resonance (MR) imaging modalities. The report includes details of preparing the methacrylated gelatin-based bioinks and the incorporation of superparamagnetic iron oxide nanoparticles. A detailed protocol is presented for characterizing the bioink, evaluating cell response, and assessing its antibacterial effect. Overall, this article presents a robust approach for the development of antibacterial, MR-visible bioinks suitable for various tissue engineering applications. For complete details on the use and execution of this protocol, please refer to Theus et al.¹.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous synthetic chemicals that threaten public health, and serum albumin binding of PFAS represents one major variable influencing PFAS toxicokinetics. In this protocol, we describe a differential scanning fluorimetry (DSF) assay suitable for the rapid determination of the relative binding affinities of serum albumin proteins to different PFAS. Herein, we address common experimental challenges related to PFAS solubility constraints, the high background fluorescence of DSF with serum albumins, and the limitations of using DSF-derived dissociation constants (K_D) to quantify PFAS-albumin interactions. For complete details on the use and execution of this protocol, please refer to Jackson et al.¹.

The development of fast ligation chemistries for the site-specific modification of proteins has become a major focus in chemical biology. We describe steps for preparing an oxalyl thioester precursor in the form of an N-oxalyl perhydro-1,2,5-dithiazepine handle, i.e., the ^oxoSEA group, and incorporating it into a peptide modifier using solid phase peptide synthesis. We then detail procedures for its application for the modification of an N-terminal Cys-containing B1 domain of the streptococcal G protein using the native chemical ligation. For complete details on the use and execution of this protocol, please refer to Snella et al.¹.

Skeletal muscle is critically dependent on the function of muscle stem cells (MuSCs) for effective muscle repair following injury. Here, we detail a protocol for the isolation of primary muscle cells and subsequent analysis of proliferation capacity in vitro using EdU (5-ethynyl-2'-deoxyuridine) on fixed cells. We also describe a cell death analysis on living cells with the identification of early- and late-apoptotic cells, as well as necrotic cells, through the incorporation of propidium iodide and YO-PRO-1 staining. For complete details on the use and execution of this protocol, please refer to Garcia et al.¹.

Liquid-liquid phase separation (LLPS) of scaffold proteins has often been proposed to drive the biogenesis of membraneless cellular compartments. Here, we present a protocol to link in vitro LLPS propensity to localization in vivo. We describe steps for examining LLPS in vitro in the presence of crowding agents or cytomimetic media. We complement our in vitro studies with recombinant proteins with experiments of protein electroporation into mitotic HeLa cells. In addition, we discuss steps to assess protein localization and delivery levels. For complete details on the use and execution of this protocol, please refer to Hedtfeld et al.¹.

Single-molecule imaging (SMI) is a powerful approach to quantify the spatiotemporal dynamics of transcription in living cells. Here, we describe a protocol of SMI for transcription and epigenetic factors in human cortical neurons derived from embryonic stem cells or induced pluripotent stem cells. Specifically, we detail the procedures for neural stem cell culture, gene transfer, microscopy, and data analysis. This protocol can be applied to the study of transcription dynamics in response to various cellular stimuli. For complete details on the use and execution of this protocol, please refer to Atsumi et al.¹.

Single-cell analysis of human peripheral blood cells provides insights into innate and adaptive immune systems. However, robust protocols are essential to ensuring single-cell sequencing data quality and cell viability. Here, we present a protocol for acquiring high-quality single-cell multi-omics data from human peripheral blood mononuclear cells (PBMCs). We describe steps for collecting human blood followed by single-cell sequencing, whole-genome sequencing, and metabolome and proteome analysis of PBMCs using modified multi-omics sample processing.