Simultaneous quantifications of protein-DNA contacts and transcriptomics in single cells (KBM7 data)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE123585 | GEO | 2019/04/21
REPOSITORIES: GEO
Similar Datasets
Project description:Here we present a new version of the DamID method that allows efficient capturing of protein-DNA interactions and mRNA output from the same single cell. With this protocol, we have measured the direct impact of spatial genome positioning and chromatin accessibility on mRNA output in mouse embryonic stem cells
2019-04-21 | GSE123586 | GEO
Project description:The series were performed to study the changes in gene expression upon diploidization of KBM7 cancer (CML) cell line. The line can exist either as a clone with 24 chromosomes (nearly haploid) or with 48 chromosomes (nearly diploid). Gene expression patterns are largely ploidy-independent, as suggested by this experiment Single cell derived clones of KBM7 cell line were grown. Included 7 haploid and 10 diploid clones, each with 2 independent total RNA extraction/microarray run. Also included peripheral blood mononuclear cells (PBMC) samples for comparison purposes.
2014-04-10 | E-GEOD-56465 | biostudies-arrayexpress
Project description:New methods that measure the static abundance of DNA, RNA, and proteins in single cells are rapidly transforming biology. But these methods do not provide insight into dynamic cellular activities, precluding a complete understanding of cellular heterogeneity. We developed a method to measure enzymatic activities in single cells and used it to simultaneously measure DNA repair enzyme activities and mRNA expression. By including polyadenylated DNA hairpin substrates with defined lesions in a single-cell mRNA sequencing experiment, we measured repair activities by capturing repair intermediates and products catalyzed by activities in single cells.
2019-10-02 | GSE130117 | GEO
Project description:The series were performed to study the changes in gene expression upon diploidization of KBM7 cancer (CML) cell line. The line can exist either as a clone with 24 chromosomes (nearly haploid) or with 48 chromosomes (nearly diploid). Gene expression patterns are largely ploidy-independent, as suggested by this experiment
2014-04-10 | GSE56465 | GEO
Project description:Simultaneous quantifications of protein-DNA contacts and transcriptomics in single cells (KBM7 data)
| PRJNA509261 | ENA
Project description:By hybridizing mRNA to oligonucleotide arrays and searching for probes that are outliers in their probe set, we identify and genotype polymorhisms in two strains of yeast (BY, isogenic to S288C, and RM, a wild vineyard strain) and segregants from a cross between the two strains. We then use this mRNA based genotyping approach to study allele-specific expression in diploid hybrids from a cross between BY and RM. A 1:1 mixture of BY and RM parental mRNA is created and hybridized to arrays as a control in these allele-specific expression experiments. The S. cerevisiae strains BY4716, an S288C derivative, and RM11-1a, a haploid Bb32(3) derivative, are described elsewhere (Brem et al. 2002, Yvert et al. 2003). We grew cultures to 10e7 cells/mL in shake flasks at 175 rpm at 300 C in synthetic C medium. We followed the standard Affymetrix protocol for preparation of RNA samples and for hybridization of the samples to Affymetrix YGS98 expression arrays. In total, three cultures of the BY strain, three cultures of the RM strain, one culture of each of the two haploid segregants and the two diploid segregants hybrids, three cultures of the BY-BY hybrid, three cultures of the RM-RM hybrid, and six cultures of the BY-RM hybrid were analyzed. Values calculated using the method described by Zhang et al. (Nat Biotechnol. 21, 818-821). The results are part of Ronald et al. (2005) describing a novel use of oligonucleotide expression arrays to perform genotyping. This method requires the use of individual probe signals, rather than the overall probeset value as is produced by analysis programs such as MAS or RMA.
2004-11-16 | GSE1975 | GEO
Project description:Many thousand long non-coding (lnc) RNAs are mapped in the human genome. Time consuming studies using reverse genetic approaches by post-transcriptional knock-down or genetic modification of the locus demonstrated diverse biological functions for a few of these transcripts. The Human Gene Trap Mutant Collection in haploid KBM7 cells is a ready-to-use tool for studying protein-coding gene function. As lncRNAs show remarkable differences in RNA biology compared to protein-coding genes, it is unclear if this gene trap collection is useful for functional analysis of lncRNAs. Here we use the uncharacterized LOC100288798 lncRNA as a model to answer this question. Using public RNA-seq data we show that LOC100288798 is ubiquitously expressed, but inefficiently spliced. The minor spliced LOC100288798 isoforms are exported to the cytoplasm, whereas the major unspliced isoform is nuclear localized. This shows that LOC100288798 RNA biology differs markedly from typical mRNAs. De novo assembly from RNA-seq data suggests that LOC100288798 extends 289kb beyond its annotated 3' end and overlaps the downstream SLC38A4 gene. Three cell lines with independent gene trap insertions in LOC100288798 were available from the KBM7 gene trap collection. RT-qPCR and RNA-seq confirmed successful lncRNA truncation and its extended length. Expression analysis from RNA-seq data shows significant deregulation of 41 protein-coding genes upon LOC100288798 truncation. Our data shows that gene trap collections in human haploid cell lines are useful tools to study lncRNAs, and identifies the previously uncharacterized LOC100288798 as a potential gene regulator.
2015-12-22 | GSE71284 | GEO
Project description:Protein-DNA interactions are critical to the regulation of gene expression, but it remains challenging to define how cell-to-cell heterogeneity in protein-DNA binding influences gene expression variability. Here we report a method for the simultaneous quantification of protein-DNA contacts by combining single-cell DNA adenine methyltransferase identification (DamID) with messenger RNA sequencing of the same cell (scDam&T-seq). We apply scDam&T-seq to reveal how genome-lamina contacts or chromatin accessibility correlate with gene expression in individual cells. Furthermore, we provide single-cell genome-wide interaction data on a polycomb-group protein, RING1B, and the associated transcriptome. Our results show that scDam&T-seq is sensitive enough to distinguish mouse embryonic stem cells cultured under different conditions and their different chromatin landscapes. Our method will enable the analysis of protein-mediated mechanisms that regulate cell-type-specific transcriptional programs in heterogeneous tissues.
| S-EPMC6609448 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2019-04-21 | GSE108639 | GEO
Project description:Background: DNA in the nucleus of a living cell carries out its functions in the context of a complex, three-dimensional chromatin architecture. Several recently developed methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of loci in yeast, fruit fly, human and mouse. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication. Here we describe a novel assay to map genome-wide chromatin contacts, tethered multiple 3C (TM3C), that involves a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by deep DNA paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. Results: We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, as well as a low noise-to-signal ratio between inter- and intrachromosomal contacts. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Ph+ formation in KBM7. Furthermore, we develop a two-phase mapping strategy that separately maps chimeric subsequences within a single read, allowing us to identify contacts involving three or four loci simultaneously, potentially corresponding to combinatorial regulation events. This mapping strategy also greatly increases the number of distinct binary contacts identified and, therefore, the coverage obtained for a fixed number of mapped reads. We confirm a subset of the triplet contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Assaying the genome architecture of a near-haploid cell line allows us to create 3D models of a human cell line without averaging signal from two homologous copies of a chromosome. Our 3D models of KBM7 show clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines. Conclusion: TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and complex chromatin loops that may play a role in regulating reciprocal expression of the IGF2 and H19 genes. Analysis of the spatial organization of two human cell lines (KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line) using tethered multiple 3C (TM3C), a novel and simple protocol for ascertaining genome architecture which can be used to identify simultaneous contacts among three or four loci in addition to binary contacts that can be identified using traditional chromosome conformation capture coupled with next generation sequencing (Hi-C).
2015-04-29 | E-GEOD-60623 | biostudies-arrayexpress