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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

NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

Project description:The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. To better map transcription genome-wide at base pair resolution and with transcription/elongation factor dependency we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). NET-prism introduces an immunoprecipitation to capture RNA Pol II – associated proteins, which reveals the interaction of these proteins with active RNA Pol II. Application of NET-prism on different Pol II variants (Pol II S2ph, Pol II S5ph), elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals diverse Pol II signals, at a single nucleotide resolution, with regards to directionality and intensity over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity.

2019-07-02 | GSE107257 | 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

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

RNA Polymerase II transcription independent of TBP [NET-seq]

Project description:Using NET-seq to profile nascent RNA transcription, we show that auxin-mediated depletion of TBP does not affect Pol II transcription and gene activation via heat shock.

2023-04-12 | GSE206695 | GEO

Investigating the role of Hmo1 in transcription by Pol I in yeast.

Project description:The purpose of this study was to investigate the role of the yeast protein, high mobility group protein (Hmo1), in rRNA synthesis. For over twenty years, Hmo1 has been proposed to be an activator of transcription by Pol I, but its regulatory mechanism remains largely undefined. Therefore, we used native elongating transcript sequencing (NET-seq) to explore the role of this factor in transcription by Pol I. Our NET-seq results demonstrated that in cells lacking Hmo1 (hmo1D), Pol I occupancy was significantly increased across the rDNA template, indicating an increased pause propensity in these cells. These data suggest that Hmo1 is an important transcription elongation factor for Pol I.

2023-11-20 | GSE247981 | GEO

Net Cleaning

Project description:Net Cleaning Raw sequence reads

| PRJNA930680 | ENA

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

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

| PRJNA553775 | ENA

Coordination of widespread transcription at enhancers and target genes through BRD4 (His-NET-Seq)

Project description:Gene transcription by RNA polymerase II (Pol II) is under the control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II which correlates with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcriptional enhancers in human cells. An integrated multi-omics analysis shows that transcription at most enhancers depends on the BET protein BRD4. We provide evidence for a direct BRD4-mediated coupling of Pol II transcription at enhancers and target genes. Specifically, BRD4 links elongation activation at enhancers and genes by maintaining their proximity. These studies reveal that transcription at enhancers and target genes is coordinated at the level of elongation.

2023-07-06 | GSE214593 | GEO

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