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Testing the usage of divalent metal cations and nucleases during NET-seq for Pol I

ABSTRACT: Testing the usage of divalent metal cations and nucleases during NET-seq for Pol I

PROVIDER: PRJNA893665 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22028413.fastq.gz	Fastqsanger.gz
	SRR22028414.fastq.gz	Fastqsanger.gz
	SRR22028415.fastq.gz	Fastqsanger.gz
	SRR22028416.fastq.gz	Fastqsanger.gz
	SRR22028417.fastq.gz	Fastqsanger.gz

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Similar Datasets

Testing the usage of divalent metal cations and nucleases during NET-seq for Pol I

Project description:The goal of this study was to determine the effects of commonly used nuclease treatments and their divalent metal cation cofactors on Pol I occupancy during NET-seq. Other groups have previously used micrococcal nuclease (MNase) + CaCl2 and deoxyribonuclease I (DNase I) + MnCl2 in NET-seq protocols probing for Pol II occupancy. However, the effect of these treatments on polymerase occupancy has not been fully explored. Therefore, we tested the impact of these treatments (CaCl2, CaCl2 + MNase, MnCl2, and MnCl2 + DNase I) on Pol I occupancy in vivo. We found that the exposure of Pol I to either CaCl2 or MnCl2 caused a significant reduction in occupancy on the rDNA template. Nuclease treatment (with either MNase or DNase I) did not cause an additional significant effect on Pol I occupancy in vivo beyond that of either CaCl2 or MnCl2. Finally, we found that MnCl2 treatment caused a more significant reduction in Pol I occupancy as compared to CaCl2, suggesting that perhaps MnCl2 stimulates the intrinsic cleavage activity of Pol I more robustly vs. CaCl2 in vivo. Altogether, these findings indicate that the inclusion of these treatments (especially that of divalent metal cations) in NET-seq protocols for Pol I cause a significant change in the polymerase occupancy.

2022-10-27 | GSE216460 | GEO

The effect of metal cations on the capture of Pol II-derived nascent transcripts in NET-seq experiments

Project description:The goal of this study was to test the effect of metal cations (CaCl2 and MnCl2) during Pol II NET-seq. These metals are readily used in the majority of studies that use NET-seq to analyze Pol II occupancy, but their effect on nascent transcript capture has not been analyzed. Our results suggest that the inclusion of these metals in Pol II NET-seq experiments does not cause a significant change in Pol II occupancy in the untreated control vs. metal-treated (CaCl2 or MnCl2) samples.

2024-05-13 | GSE267082 | GEO

The effect of metal cations on the capture of Pol II-derived nascent transcripts in NET-seq experiments

Project description:The effect of metal cations on the capture of Pol II-derived nascent transcripts in NET-seq experiments

| PRJNA1109721 | ENA

Net Cleaning

Project description:Net Cleaning Raw sequence reads

| PRJNA930680 | ENA

Randomized PAM depletion of phylogenetically-diverse Cas12a nucleases

Project description:We report the PAMs of phylogenetically-diverse Cas12a nucleases using a cell-free TXTL-based PAM screen. By adding a 5N randomized PAM library and Cas12a-gRNA in vitro, recognized PAM sequences were cleaved, while non-recognized PAMs remained. By amplifying the non-cleaved DNA, we used next-generation sequencing to analyze the depletion of functional PAMs of these Cas12a nucleases.

2020-03-02 | GSE130377 | GEO

METAL RUST

Project description:METAL RUST Metagenome

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Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Project description:Because of their smallness, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.

2022-09-12 | GSE213149 | GEO

NET-seq analysis of pSer2-Pol II profile in shCTRL, shMETTL3 and shDDX21 HeLa cells

Project description:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was puriﬁed with SpinX column (Coster) with 2 glass ﬁlters (Millipore) and the ﬂow-through RNA was ethanol precipitated.

2019-10-15 | GSE134102 | GEO

Expression profile of Gastro-Entero-Pancreatic Neuroendocrine Tumors (GEP-NET)

Project description:Expression profile of human GEP-NET tumors, including 113 fresh frozen biopsies of primary and metastatic tumours originating from pancreas (P-NET, 83 primary and 30 metastasis), 81 from small intestine (SI-NET, 44 primary and 37 metastasis), and 18 from rectum (RE-NET, 3 primary and 15 metastasis).

2018-02-25 | GSE98894 | GEO

Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

Project description:We describe a robust linear amplification-mediated high-throughput genome-wide translocation sequencing (HTGTS) method that identifies endogenous or ectopic "prey" DNA double-stranded breaks (DSBs) across the human genome based on their translocation to "bait" DSBs generated by engineered nucleases. HTGTS with different Cas9:gRNA or TALEN-nuclease on-target baits revealed off-target hotspots for given nucleases that ranged from few or none to dozens or more, and greatly extended known off-target numbers for certain previously characterized engineered nucleases by more than 10-fold. Beyond various types of nuclease off-target collateral damage, we also identified collateral damage in the form of translocations between bona fide nuclease targets on homologous chromosomes. Based on frequent non-specific DSBs making any given human chromosome an HTGTS hotspot region for bait DSBs within it, we found that HTGTS also reveals wide-spread, low-level DSB-generating activities of engineered nucleases. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-targets genome-wide and suggested other strategies to enhance desired nuclease activities.

2014-11-29 | GSE57283 | GEO

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