STAR Protocols最新文献

Gene knockdown by small interfering RNA (siRNA) transfection is widely used to investigate gene function. Here, we present a protocol for the knockdown of the odorant-binding protein 2A (OBP2A) gene in a three-dimensional human epidermal equivalent model (3DE-model). We describe steps for growing human epidermal keratinocytes (normal human epithelial keratinocytes [NHEKs]) for 3DE-model construction in monolayer culture. We then detail procedures for transfecting the cells with siRNA, followed by a second siRNA transfection during 3DE-model construction. The efficiency of gene knockdown is verified by qPCR and ELISA. For complete details on the use and execution of this protocol, please refer to Nakanishi et al.¹.

Lactic acid bacteria (LAB) are a large group produced during carbohydrate fermentation, resulting in lactic acid. Here, we present a protocol for probiotic adhesion and interaction into buccal epithelial cells, chicken epithelial cells, and HT-29 cells, as well as for autoaggregation and coaggregation. We describe steps for visualizing LAB adhesion by imaging cell adhesion using a light microscope. We then detail procedures for performing an adhesion assay. For complete details on the use and execution of this protocol, please refer to Kumari et al.¹.

Plant m⁶A modification is highly dynamic, with plant hormones serving as key regulators that induce these dynamics. Here, we present a protocol for detecting phytohormone-induced changes in m⁶A modification using RNA dot blot and m⁶A sequencing (m⁶A-seq) techniques. We describe steps for the auxin treatment, RNA dot blot and m⁶A-seq library preparation, and data analysis. This protocol holds potential applications for analyzing changes in m⁶A modification induced by plant hormones. For complete details on the use and execution of this protocol, please refer to Bin et al.¹.

Spatially resolved transcriptomics (SRT) data contain intricate noise due to the diffusion of transcripts caused by tissue fixation, permeabilization, and cell lysis during the experiment. Here, we present a protocol for denoising SRT data using SpotGF, an optimal transport-based gene filtering algorithm, without modifying the raw gene expression. We describe steps for data preparation, SpotGF score calculation, filtering threshold determination, denoised data generation, and visualization. Our protocol enhances SRT quality and improves the performance of downstream analyses. For complete details on the use and execution of this protocol, please refer to Du et al.¹.

Here, we present a protocol for the quantitative characterization of human T cell aging. We describe steps for sample collection; peripheral blood mononuclear cell (PBMC) isolation; and the enrichment, assessment, and activation of naive CD8⁺ T cells. We then detail procedures for supernatant collection and quantification using the absolute copy number of mitochondrial DNA (mtDNA) released into the supernatants from in-vitro-activated naive CD8⁺ T cells. We also apply this approach to predict the occurrence of lung adenocarcinoma in middle-aged populations. For complete details on the use and execution of this protocol, please refer to Jin et al.¹.

RNA 5-methylcytosine (m⁵C) is a widespread modification and plays a crucial role in gene expression regulation. Here, we present a protocol for transcriptome-wide m⁵C methylome profiling at base resolution using bisulfite-free m⁵C detection strategy enabled by ten-eleven translocation (TET)-assisted chemical labeling sequencing (m⁵C-TAC-seq). We detail steps for TET-assisted chemical labeling, library construction, and data analysis. m⁵C-TAC-seq enables accurate and robust m⁵C detection in various RNA species. For complete details on the use and execution of this protocol, please refer to Lu et al.¹.

Killer yeast produce antimicrobial proteins, often together with a self-protective immunity factor. Here, we present a protocol for analyzing the toxic, immune, and suicidal phenotypes in Saccharomyces cerevisiae. We describe steps for assessing toxicity via halo assays, suicidality through spot assays, and immunity using microtiter plate growth assays. We detail procedures for yeast culturing, performing the assays, and data analysis. For complete details on the use and execution of this protocol, please refer to Prins et al.¹.

Analyzing whole-genome sequencing (WGS) data from bacterial isolates is pivotal for understanding virulence and predicting clinical outcomes through association studies. Herein, we present a computational protocol for the detailed analysis of WGS data from Staphylococcus aureus clinical isolates generated with Illumina sequencing. We describe steps for de novo assembly, functional annotation, and genetic characterization of chromosomal and extrachromosomal elements. This approach paves the way for an improved understanding of the interplay between virulence factors, resistome, strain type, and disease severity. For complete details on the use and execution of this protocol, please refer to Sánchez-Osuna et al.¹.

Extended pluripotent stem cells (EPSCs) possess a high differentiation capacity, potentially as a superior seed resource for generating cardiomyocytes. Here, we present a protocol for generating feeder-free EPSCs (ffEPSCs), cardiomyocytes, and engineered heart tissues (EHTs). We describe steps for converting human embryonic stem cells or induced pluripotent stem cells (ESCs/iPSCs) into ffEPSCs, followed by their long-term maintenance, cryopreservation, seed preservation, and differentiation into cardiomyocytes. We then detail procedures for constructing and culturing three-dimensional EHTs followed by their contraction force measurement and optical mapping. For complete details on the use and execution of the protocol, please refer to Zheng et al.¹ and Li et al.².

Protein import into the mitochondria is required for organellar function. Inefficient import can result in the stalling of mitochondrial precursors inside the translocase of the outer membrane (TOM) and blockage of the mitochondrial entry gate. Here, we present a protocol to assess the clogging of TOM by mitochondrial precursors in human cell lines. We describe how the localization of mitochondrial precursors can be determined by cellular fractionation. We then show how co-immunoprecipitation can be used to test the stalling of precursors inside TOM. For complete details on the use and execution of this protocol, please refer to Kim et al.¹.