Simultaneous profiling of transcriptome and chromatin accessibility in single nucleus
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE126074 | GEO | 2019/02/05
REPOSITORIES: GEO
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Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing "in-nucleus ligation" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications. We created Hi-C libraries by two different methods, in-solution ligation and in-nucleus ligation, from two biological replicates each of mouse foetal liver cells (mouse-1 and mouse-2) and human ES cells (human-1 and human-2) or the mixture of these two species. We also sequenced a random ligation library prepared by reversal of the cross-links and purification of the DNA prior to ligation.
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Project description:Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing has been used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis during tumor processing. Additionally, frozen tumor samples constitute a vast and valuable material bank for cancer research. Therefore, we developed a high-throughput and high-sensitivity method called HH-seq, which combines random primers and a pre-index strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. We also established a robust pipeline to facilitate the analysis of full-length RNA-seq data. We applied HH-seq to more than 730,000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables us to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns and clone dynamic, etc. We identified new malignant cell subclusters and explored their specific function across cancers. Furthermore, we investigated the malignant status of epithelial cells among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.
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Project description:The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome. Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice. This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.
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Project description:We present DEFND-seq (DNA and Expression From Nucleosome Depletion), a scalable method for co-sequencing RNA and DNA from single nuclei. In DEFND-seq we treat nuclei with lithium diiodosalicylate to disrupt chromatin and expose genoimc DNA. The nuclei are then tagmented with Tn5 transposase, which fragments and tags gDNA. Tagmented nuclei are loaded into a microfluidic droplet generator which combines nuclei, beads containing transcriptomic and genomic barcodes, and reverse transcription reagents into single droplets. Ultimately two libraries are created, one for nuclear mRNA and one for genomic DNA, with each library containing barcodes linking it to its nuclei of origin, thus allowing simultaneous analysis of single nuclei transcriptomes and genomes. Once nuclei have been depleted of nucleosomes, all steps can be performed using a 10x Chromium Controller and 10x Multiome Kit without further experimental modification.
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