Purification of GFP-tagged nuclei from frozen livers of INTACT mice for RNA- and ATAC-sequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Loft A
PROVIDER: S-EPMC8433282 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundThe genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.ResultsIn this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.ConclusionsWe have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.
| S-EPMC10201787 | biostudies-literature
Project description:The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.
| S-EPMC8864739 | biostudies-literature
Project description:It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.
| S-EPMC3856806 | biostudies-literature
Project description:Single-nucleus ATAC sequencing (snATAC-seq) employs a hyperactive Tn5 transposase to gain precise information about the cis-regulatory elements in specific cell types. However, the standard protocol of snATAC-seq is not optimized for all tissues, including the brain. Here, we present a modified protocol for single-nuclei isolation from postmortem frozen human brain tissue, followed by snATAC-seq library preparation and sequencing. We also describe an integrated bioinformatics analysis pipeline using an R package (ArchRtoSignac) to robustly analyze snATAC-seq data. For complete details on the use and execution of this protocol, please refer to Morabito et al. (2021).
| S-EPMC9218237 | biostudies-literature
Project description:An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000-33,000 accessible chromatin regions in lamina and 22-61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments.
| S-EPMC8242358 | biostudies-literature
Project description:Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA-protein complexes in a wide range of bacteria.
| S-EPMC2777422 | biostudies-literature
Project description:The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.
| S-EPMC3912835 | biostudies-other
Project description:We report the application of different chromatin profiling techniques applied to cell-type specific nuclei obtained with a nuclei immuno-enrichment protocol described in this manuscript, referred as NEI protocol. By optimizing these methods, we generated genome-wide datasets associated with the nuclear RNA transcriptome, chromatin accessibility, and distribution of histone H3 and two H3 two lysine tri-methylation marks, H3K4me3 and H3K36me3 respectively. This study provides a framework for the application of different aspects of chromatin biology using nuclei derived from specific cell types in Drosophila. Further, it demonstrates the low input requirements necessary for these chromatin studies.
2021-05-23 | GSE169328 | GEO
Project description:Background:Organ transplantation is considered the best treatment for end-stage organ failure. However, the lack of available organs for transplantation and the increasing number of patients waiting for transplants are primary issues facing the transplant community. Thus, developing strategies to increase the number of donors, especially for liver transplantation, has become a priority. The use of organs acquired from donors who suffered cardiac related deaths has increased the pool of potential liver donors. However, donation after cardiac death (DCD) livers increases the risk of primary graft dysfunction. Methods:In the current study, we conducted transcriptome sequencing using livers from a DCD rat to assess the short-term feasibility and functional efficacy of DCD livers. RNA sequencing (RNAseq) data showed that the liver transcriptome varied greatly in rat livers subjected to 15 minutes of cardiac arrest. Results:The livers used in the current study had a significant loss of normal function before transplantation. Functional and network analyses consistently indicated that transcription and translation processes were inhibited after approximately 15 minutes of cardiac arrest. Moreover, the transcriptomic sequencing data provides significant insight for identifying functional genes and testing additional biological questions in DCD liver transplantation in future studies.
| S-EPMC6241343 | biostudies-literature
Project description:The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the macrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP-expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed.
| S-EPMC3013146 | biostudies-literature