STAR Protocols最新文献

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.

Complex coacervate droplets are synthetic cell models that sequester nucleic acids. Here, we present a protocol for the recovery and deep sequencing of single-stranded DNA (ssDNA) from metabolically active coacervate droplets. We describe time-resolved harvesting, ssDNA recovery, and sequence-independent library preparation for next generation sequencing (NGS), along with an analysis pipeline to assess enrichment dynamics and sequence distributions. The protocol is adaptable to diverse droplet systems. For complete details on the use and execution of this protocol, please refer to Machatzke et al.¹.

Selected genes are genomic regions shaped by selection pressure and are often associated with important agronomic traits. Here, we present a protocol for identifying selected genes using genome resequencing data, followed by haplotype analysis of these genes. We describe steps for sequencing data collection and preprocessing, detection of genomic regions under selection, and haplotype construction based on sequence variation. The selected genes and haplotypes identified using this protocol provide insights into the genetic basis of soybean adaptation and improvement. For complete details on the use and execution of this protocol, please refer to Zhu et al.¹.

The absence of reliable biomarkers impedes the objective diagnosis and monitoring of many psychiatric symptoms. Here, using cue-induced craving in methamphetamine use disorder as a case example, we present a protocol for linking neurophysiological characteristics to clinical symptoms through high-density electroencephalography (HD-EEG). We describe steps for HD-EEG data acquisition, signal preprocessing, source localization, and functional connectivity network construction. We then detail procedures for association analysis with clinical symptom scores. This protocol holds potential for application to diverse psychiatric conditions. For complete details on the use and execution of this protocol, please refer to Tian et al.¹.

Identification of integration sites of human immunodeficiency virus (HIV) is crucial for developing antiretroviral strategies. Here, we present an in vitro transcription-based version of lentiviral integration site sequencing (LIS-seq), enabling rapid integration site mapping. We detail steps for the establishment of clonal cellular models, genomic DNA isolation and fragmentation, T7 in vitro transcription, poly(A) tailing, sequencing library generation, and the analytical pipeline. LIS-seq has been applied to clonal cellular models infected with an HIV-based vector. For complete details on the use and execution of this protocol, please refer to Więcek et al.¹.

Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers. For complete details on the use and execution of this protocol, please refer to Grissom et al.¹.

Isolating primary dermal fibroblasts is often costly, time-consuming, has high contamination risk, and is inefficient, yielding low cell numbers and requiring long culture periods. Here, we present a non-enzymatic isolation protocol from mouse skin that overcomes these limitations and consistently provides passage 1 (P1) primary fibroblasts with every tissue subculture. We describe steps for isolating primary fibroblasts from adult mice dermis by placing a precise small dermal tissue layer, which adhered after 24 h, with migrating fibroblasts observed at 48 h post isolation.

Single-molecule analysis of replicated DNA (SMARD) is a powerful tool to study DNA replication by visualizing de novo-incorporated nucleotide analogs in individual DNA molecules. Here, we present the protocol to investigate replication kinetics of Kaposi's sarcoma-associated herpesvirus using SMARD. We describe steps for seeding cells, nucleotide analog pulsing of cells, preparation and digestion of agarose plugs followed by pulsed-field gel electrophoresis, probe labeling, DNA stretching, and fluorescence imaging to map replication origins and fork progression. For complete details on the use and execution of this protocol, please refer to Verma et al.¹ and Singh et al.².