STAR Protocols最新文献

Tumor-specific DNA methylation profiling in plasma cell-free DNA (cfDNA) offers a promising approach for non-invasive tumor detection. Here, we present a protocol that uses Oncoder, an interpretable deep-learning-based framework, to monitor treatment response by tracking dynamic changes in tumor-specific DNA methylation signals in patient plasma cfDNA. We describe steps for data preparation, performing differential methylation analysis, training Oncoder, and interpreting the model's outputs. This protocol is versatile and adaptable to various data types and application scenarios. For complete details on the use and execution of this protocol, please refer to Yang et al.¹.

Transfer RNA (tRNA), its post-transcriptional modifications, and tRNA-derived fragments (tRFs) play essential roles in cellular processes and gene regulation. Here, we present a fast and efficient tRNA enrichment protocol using silica spin columns. To analyze tRNA and tRFs, we describe steps for DNA probe labeling, tRNA separation on polyacrylamide (PAA) gels, and RNA transfer, UV crosslinking, and non-radioactive northern blotting. Additionally, this protocol describes the use of chemical affinity modifiers, enabling the detection of chemical modifications in specific tRNA isoacceptors.

Here, we present a protocol to simultaneously release and profile N- and O-glycans from glycoproteins in a one-pot format. We describe steps for protein reduction, alkylation, N-glycan release, Proteinase K digestion, HILIC enrichment, N-glycan reduction, N-/O-glycan permethylation while releasing O-glycans, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). For complete details on the use and execution of this protocol, please refer to Ortega-Rodriguez et al.¹.

CRISPR-virus-like particle (VLP)-induced targeted mutagenesis (CRISPR-VIM) enables genome editing in mouse embryos through non-invasive delivery of CRISPR ribonucleoproteins (RNPs) via VLPs, eliminating the need for physical manipulation and specialized expertise. We detail protocols for VLP production, titration, and treatment for diverse genome edits. This protocol is compatible with zygotes and in vitro fertilization (IVF)-derived embryos via simple co-culture, facilitating high-efficiency and heritable mutations with minimized off-target effects, independent of specialized equipment and conducive to reduced animal use. For complete details on the use and execution of this protocol, please refer to Jeong et al.¹.

Most developmental processes underpinning the diversity of floral forms remain unexplored due to limitations in current imaging approaches. Here, we present a protocol to spatially analyze tissue-wide cell growth dynamics through confocal imaging and segmentation of the adaxial petal epidermis in Hibiscus trionum. We describe steps for dissecting buds, staining petals with a membrane dye, and acquiring images suitable for epidermal reconstruction. We detail procedures for image stitching and processing in ImageJ and for surface extraction and cell segmentation in MorphoGraphX. For complete details on the use and execution of this protocol, please refer to Riglet et al.¹.

Comprehensive immune profiling of human blood is a critical approach for evaluating immune responses following therapeutic intervention in clinical trials. Here, we present a protocol to assess leukocyte lineages, activation status, and selected immune checkpoint expression in cryopreserved buffy coat using a high-dimensional, 49-color spectral flow cytometry antibody panel. We provide instructions for thawing, washing, and staining circulating immune cells, followed by guidance on data acquisition. This protocol is suitable for studying cancer immunology and other indications requiring deep immunophenotyping.

Characterizing cell proliferation across developing tissue provides critical insights into how spatially regulated growth shapes mature organs. Here, we present a protocol for high-resolution mapping of cell division events across the epidermis of Hibiscus trionum petals. We outline steps for labeling newly replicated DNA through incubation in EdU-containing medium, combined with floral bud dissection and confocal imaging. We also detail procedures for sample clearing and labeling of nuclei and cell outlines to enable accurate quantification of division patterns. For complete details on the use and execution of this protocol, please refer to Riglet et al.¹.

The brain, specifically the hypothalamus, is a key regulator of the homeostatic balance of numerous biomolecules in the body via its control of the neuroendocrine system. Here, we present a protocol to study body vitamin A balance by injecting synthetic retinoids into the third ventricle of the rat brain. We also describe steps for the intraventricular application of viral vectors to the arcuate nucleus of a mouse brain to knock down vitamin A homeostatic genes. This approach is applicable to the study of vitamin A homeostasis. For complete details on the use and execution of this protocol, please refer to Imoesi et al.¹.

Complementary metal-oxide-semiconductor (CMOS)-based high-density microelectrode arrays (HD-MEAs) enable neuronal recordings with high spatiotemporal resolution. However, integrating polydimethylsiloxane (PDMS) microstructures onto HD-MEA surfaces to control network architecture is currently challenging and platform specific. Here, we present a protocol for PDMS fabrication, HD-MEA chip preparation, PDMS-HD-MEA microstructure alignment, and cell culture, including alternatives. Our results show reproducible formation of modular networks with characteristic activity patterns across different systems. This protocol supports engineering of defined neuronal architectures while maintaining compatibility with various HD-MEA systems. For complete details on the use and execution of this protocol, please refer to Sato et al.¹.