The DNA cleavage efficiency of the hpCasMINI system [Deep-seq]
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ABSTRACT:
PROVIDER: PRJNA1127531 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The fusion of an α-helix to the N-terminus of CasMINI (hpCasMINI) could indeed enhance the DNA cleavage efficiency in mammalian cells.
2024-06-29 | GSE270587 | GEO
Project description:We perform polyA independent deep sequencing of chromatin associated primary transcripts across three different cell lines to obtain a global view on in vivo microRNA processing. We use these data to define a MicroProcessing Index (MPI), to quantify the cleavage efficiency of the Microprocessor complex. Hallmarks of efficient Drosha-mediated processing are confirmed by means of deep sequencing of chromatin-associated transcripts upon Drosha knockdown. Our results suggest that both sequence features and thermodynamic properties, e.g. secondary structure of the regions flanking the pre-miRNA hairpins are determinants for efficient processing. Our data furthermore enables us to observe endogenous microprocessor cleavage sites at nucleotide resolution. This analysis reveals the presence of non-canonical processing events occurring one helical turn distal of most efficiently cleaved miRNA precursors. We performed polyA independent deep sequencing of the chromatin-isolated RNA fraction for 5 samples: 2 replicates in HeLa cells, 1 Drosha knock down in HeLa cells, 1 sample for A549 cells and 1 sample for HEK293 cells. We also performed deep sequencing of the small RNA fraction in the same cell lines.
2014-04-17 | E-GEOD-56862 | biostudies-arrayexpress
Project description:MiRNA-mediated regulation depends on the stoichiometry between miRNAs and their mRNA targets. To decipher dynamic function of this complex layer, it is critical to characterize individual miRNA species within a specific cellular context. Small RNA cloning followed by deep sequencing is uniquely positioned as a genome-wide profiling method to quantify miRNA expression with potentially unlimited dynamic range and provide single-nucleotide resolution for precise miRNA classification and de novo discovery. However, significant biases introduced by RNA ligation steps in the current RNA cloning protocol often lead to inaccurate miRNA quantification by >1000-fold deviation. As a result, it has greatly hindered the broad application of this method. Here we report a highly efficient RNA cloning method that achieves over 90% efficiency for both 5’ and 3’ ligations with diverse small RNA substrates. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, the deviation of the cloning frequency for each miRNA is minimized to less than 2-fold of the anticipated value. By using samples obtained from multiple tissues and cells, we further demonstrate the accurate quantification of miRNA expression over a dynamic range of four orders of magnitude. Our results also reveal that most cistronic miRNAs are expressed at similar levels and, in each cell population, miRNAs repress their cognate targets in a dosage dependent manner. Collectively, our high-efficiency RNA cloning method combining with deep sequencing establishes a cost-effective approach for accurate genome-wide miRNA profiling. We designed an artificial system composed of synthetic miRNAs for benchmarking biases in small RNA cDNA cloning for NGS.
2013-09-18 | E-GEOD-47858 | biostudies-arrayexpress
Project description:Type II topoisomerases orchestrate proper DNA topology and they also are the targets of anticancer drugs that cause leukemias with balanced translocations as secondary cancers. Here, we develop a novel high-throughput sequencing technology to define TOP2 cleavage sites at single base precision in human cells and use the technology to characterize TOP2A cleavage genome-wide in the K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence predilections, occurs in cleavage cluster regions (CCRs) and is enriched in introns and lincRNA loci. In coding genes we discover a bias of TOP2A CCRs towards the distal regions of gene bodies and proximal shifts in TOP2A CCRs with TOP2 poisons. We find high TOP2A cleavage levels in the genes involved in translocations associated with TOP2 poisons in leukemia. In addition, we find that large a proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. By making comparisons to ENCODE data, we uncover distinct TOP2A CCR clusters that overlap with marks of gene transcription, open chromatin and enhancers. Our findings implicate TOP2A as the DNA damage mediator in oncogenic translocations more broadly than was previously appreciated. They also identify TOP2A as a functional DNA element that contributes to the regulation of transcription elongation and gene activation.
2017-03-08 | GSE79593 | GEO
Project description:Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854hsdM exhibited a 5-fold increase in transformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.
2021-04-14 | GSE167217 | GEO
Project description:Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g., quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50 fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e., negative vs. positive supercoiling. SHAN-seq’s exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.
2024-08-06 | GSE267149 | GEO
Project description:Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3' region extraction and deep sequencing (3'READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3' ends revealed over 5,000 previously overlooked pAs (~8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3'READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3' endM-bM-^@M-^Sprocessing activity in development and differentiation. 3'READS to map pAs in mouse genome
2012-12-17 | E-GEOD-42398 | biostudies-arrayexpress
Project description:MiRNA-mediated regulation depends on the stoichiometry between miRNAs and their mRNA targets. To decipher dynamic function of this complex layer, it is critical to characterize individual miRNA species within a specific cellular context. Small RNA cloning followed by deep sequencing is uniquely positioned as a genome-wide profiling method to quantify miRNA expression with potentially unlimited dynamic range and provide single-nucleotide resolution for precise miRNA classification and de novo discovery. However, significant biases introduced by RNA ligation steps in the current RNA cloning protocol often lead to inaccurate miRNA quantification by >1000-fold deviation. As a result, it has greatly hindered the broad application of this method. Here we report a highly efficient RNA cloning method that achieves over 90% efficiency for both 5’ and 3’ ligations with diverse small RNA substrates. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, the deviation of the cloning frequency for each miRNA is minimized to less than 2-fold of the anticipated value. By using samples obtained from multiple tissues and cells, we further demonstrate the accurate quantification of miRNA expression over a dynamic range of four orders of magnitude. Our results also reveal that most cistronic miRNAs are expressed at similar levels and, in each cell population, miRNAs repress their cognate targets in a dosage dependent manner. Collectively, our high-efficiency RNA cloning method combining with deep sequencing establishes a cost-effective approach for accurate genome-wide miRNA profiling.
2013-09-18 | GSE47858 | GEO
Project description:Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. However, it is not always clear whether specific targets of orthologous RNAse toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here we show that the TAS HigBA can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigBâs mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks. DNA binding of the antitoxin HigA and the SOS regulator LexA was analysed by chromatin immunoprecipitation-deep sequencing, and found to overlap at only one locus, the HigBA TA system promoter
2016-02-17 | E-GEOD-76721 | biostudies-arrayexpress
Project description:Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem (hiPS) cells. hiPS cells generated with this system were molecularly and functionally similar to human embryonic stem (hES) cells, demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes, suggesting that the kinetics of reprogramming are cell-type dependent. Using our inducible system, we developed a strategy to induce hiPS cell formation at high frequency by generating differentiated cells that contain the viral transgenes in a pattern that enables successful induction of pluripotency. Upon addition of doxycycline to differentiated hiPS-derived cells, we obtained “secondary” hiPS cells at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells with high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.
2008-09-12 | GSE12390 | GEO