Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes
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PROVIDER: PRJNA336549 | ENA |
REPOSITORIES: ENA
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Project description:Chromatin structure plays a pivotal role in facilitating proper control of gene expression. The ability of transcription factors (TF) to bind cis-elements is often associated with accessible chromatin regions. Therefore, identification of these accessible regions throughout plant genomes is important to understanding the relationship between TF binding, chromatin status and the regulation of gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a recently developed technique used to map open chromatin zones in animal genomes. However, in plants, the existence of cell walls, subcellular organelles and the lack of stable cell lines have prevented routine application of this technique. Here, we describe an assay combining ATAC-seq with Fluorescence-Activated Nuclei Sorting (FANS) to identify and map open chromatin and TF-binding sites in plant genomes. FANS-ATAC-seq compares favorably with published DNaseI sequencing (DNase-seq) results and it only requires 500-50,000 nuclei for accurate identification of open chromatin states.
2016-11-30 | GSE85203 | GEO
Project description:Chromatin structure plays a pivotal role in facilitating proper control of gene expression. Transcription factor (TF) binding of cis-elements is often associated with accessible chromatin regions. Therefore, the ability to identify these accessible regions throughout plant genomes will advance understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a recently developed technique used to map open chromatin zones in animal genomes. However, in plants, the existence of cell walls, subcellular organelles and the lack of stable cell lines have prevented routine application of this technique. Here, we describe an assay combining ATAC-seq with fluorescence-activated nuclei sorting (FANS) to identify and map open chromatin and TF-binding sites in plant genomes. FANS-ATAC-seq compares favorably with published DNaseI sequencing (DNase-seq) results and it requires less than 50 000 nuclei for accurate identification of accessible genomic regions.SummaryApplication of ATAC-seq to sorted nuclei identifies accessible regions genome-wide.
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Project description:Variable tumor cellularity can limit sensitivity and precision in comparative genomics, because differences in tumor content can result in misclassifying truncal mutations as region-specific private mutations in stroma-rich regions, especially when studying tissue specimens of mediocre tumor cellularity such as LUADs. To address this issue, we refined a nuclei flow-sorting approach by sorting nuclei based on ploidy and the LUAD lineage marker thyroid transcription factor 1 (TTF-1) and applied this method to investigate genome-wide somatic copy number aberrations (SCNA) and mutations of 409 cancer genes in 39 tumor populations obtained from 16 primary tumors and 21 matched metastases. This approach increased the mean tumor purity from 54% (range: 7-89%) of unsorted material to 92% (range: 79-99%) after sorting. Despite this rise in tumor purity, we detected limited genetic heterogeneity between primary tumors and their metastases. In fact, 88% of SCNAs and 80% of mutations were propagated from primary tumors to metastases and low allele frequency mutations accounted for much of the mutational heterogeneity. Even though the presence of SCNAs indicated a history of chromosomal instability (CIN) in all tumors, metastases did not have more SCNAs than primary tumors. Moreover, tumors with biallelic TP53 or ATM mutations had high numbers of SCNAs, yet they were associated with a low interlesional genetic heterogeneity. The results of our study thus provide evidence that most macroevolutionary events occur in primary tumors before metastatic dissemination and advocate for a limited degree of CIN over time and space in this cohort of LUADs. Sampling of primary tumors thus may suffice to detect most mutations and SCNAs. In addition, metastases but not primary tumors had seeded additional metastases in three of four patients; this provides a genomic rational for surgical treatment of such oligometastatic LUADs.
2018-09-11 | GSE119763 | GEO
Project description:Investigation of recombination at the DNA sequence level by combining flow-sorting of haploid pollen nuclei of barley with single-cell genome sequencing.
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Project description:Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissociation might alter gene expressions. In this work, we cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from human pancreata. We innovated fluorescence-activated nuclei sorting (FANS) based on the positive signal of NKX2-2 antibody to enrich for nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. We observed comparable endocrine cellular composition and cell type signature gene expression between our snRNA-seq libraries and conventional scRNA-seq libraries generated with live cells from freshly isolated human islets. Our work fills a technological gap and helps to unleash archival pancreatic tissue for molecular profiling targeting the endocrine population. We expect that our protocol can be used to enrich nuclei for transcriptomics study from various populations in the pancreas and in different organs/tissues.
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Project description:We report RNA-seq of single nuclei isolated from the adult C57BL/6 male mouse Hippocampus region. Majority of the nuclei were isolated from 12 weeks old mice (4 different animal), with an additional set of nuclei from 3 months and 2 years old animals. In addition a set of GFP labeled nuclei driven by a VGAT promoter . Microdissections of dentate gyrus, CA1 and CA2/3 regions of the Hippocampus were placed into ice-cold RNA-later for fixation and stored at 4âc overnight, then stored in -80âc. Nuclei were isolated by sucrose gradient centrifugation and kept on ice until sorting using Fluorescence Activated Cell Sorting (FACS) into 96 well plates containing RNA lysis buffer. Single nucleus RNA was first purified then derived cDNA libraries were generated following a modified Smart-seq2 protocol. For VGAT nuclei: high titer AAV1/2 of pAAV-EF1a-DIO-EYFP-KASH-WPRE-hGH-polyA was injected into dorsal and/or ventral Hippocampus, animals were sacrificed two weeks after injections, and GFP labeled nuclei were sorted into plates and processed as described above.
2016-08-16 | E-GEOD-85721 | biostudies-arrayexpress
Project description:DNA-binding transcription factors (TFs) play a central role in transcription regulation, and computational approaches that help in elucidating complex mechanisms governing this basic biological process are of great use. In this perspective, we present the TFM-Explorer web server that is a toolbox to identify putative TF binding sites within a set of upstream regulatory sequences of genes sharing some regulatory mechanisms. TFM-Explorer finds local regions showing overrepresentation of binding sites. Accepted organisms are human, mouse, rat, chicken and drosophila. The server employs a number of features to help users to analyze their data: visualization of selected binding sites on genomic sequences, and selection of cis-regulatory modules. TFM-Explorer is available at http://bioinfo.lifl.fr/TFM.
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Project description:Caenorhabditis elegans is a major eukaryotic experimental system employed to unravel a broad range of cellular and biological processes. Despite the many advantages of C. elegans, biochemical approaches to study tissue-specific gene expression in postembryonic stages are challenging. Here we report a novel experimental approach that enables the efficient determination of tissue-enriched transcriptomes by rapidly releasing nuclei from major tissues of postembryonic animals followed by fluorescence-activated nuclei sorting (FANS). Furthermore, we developed and applied a deep sequencing method, named 3'end-seq, which is designed to examine gene expression and identify 3' ends of transcripts using a small quantity of input RNA. In agreement with intestinal specific gene expression, promoter elements of highly expressed genes are enriched for GATA elements and their functional properties are associated with processes that are characteristic for the intestine. In addition, we systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including >3,000 sites that have previously not been identified. The analysis of nuclear mRNA revealed widespread alternative polyA site use in intestinally expressed genes. We describe several novel approaches that will be of significance to the analysis of tissue specific gene expression using small quantity RNA samples from C. elegans and beyond. 3'end-seq of transcriptomes for input and sorted nuclei
2011-12-01 | E-GEOD-32165 | biostudies-arrayexpress