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Genome wide mapping of native R-loops using a targeted nuclease approach in U2OS cells after DDX41 siRNA knockdown or ActinomycinD treatment (MapR)

ABSTRACT: Genome wide mapping of native R-loops using a targeted nuclease approach in U2OS cells after DDX41 siRNA knockdown or ActinomycinD treatment (MapR)

PROVIDER: PRJNA706393 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13848162_1.fastq.gz	Fastqsanger.gz
	SRR13848162_2.fastq.gz	Fastqsanger.gz
	SRR13848163_1.fastq.gz	Fastqsanger.gz
	SRR13848163_2.fastq.gz	Fastqsanger.gz
	SRR13848164_1.fastq.gz	Fastqsanger.gz

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Genome wide mapping of native R-loops using a targeted nuclease approach in U2OS cells after DDX41 siRNA knockdown or ActinomycinD treatment (MapR)

Project description:Purpose: Investigation of genome-wide changes in R-loop levels after knockdown of DDX41 U2OS cells in comparison to a control knockdown. Method: Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2). 5*105 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 50 µl Wash Buffer containing 0.05% Digitonin. Either pA-MNase or RHΔ-MNase were added to the cells overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin, samples resuspended in 100 µl Dig-Wash-Buffer were equilibrated on ice. Activity of the MNase was triggered by adding 2mM CaCl2 to the samples for 30 minutes. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, 20 pg/ml spike-in Drosophila DNA (Active Motif)) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We were able to map R-loop dynamics genome-wide in U2OS cells in biological triplicates. MapR signal was significantly increased around the transcription start site in the absence of DDX41 and decreased after treatment with ActinomycinD.

2021-05-04 | GSE168170 | GEO

Genome wide mapping of native R-loops using a targeted nuclease approach in HCT116 cells after DDX41 siRNA knockdown (MapR)

Project description:Genome wide mapping of native R-loops using a targeted nuclease approach in HCT116 cells after DDX41 siRNA knockdown (MapR)

| PRJNA759129 | ENA

Genome wide mapping of native R-loops using a targeted nuclease approach in HCT116 cells after DDX41 siRNA knockdown (MapR)

Project description:Purpose: Investigation of genome-wide changes in R-loop levels after knockdown of DDX41 HCT116 cells in comparison to a control knockdown. Method: Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2). 1*10^6HCT116 cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 50 µl Wash Buffer containing 0.05% Digitonin. Either pA-MNase or RHΔ-MNase were added to the cells overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin, samples resuspended in 100 µl Dig-Wash-Buffer were equilibrated on ice. Activity of the MNase was triggered by adding 2mM CaCl2 to the samples for 30 minutes. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, 20 pg/ml spike-in Drosophila DNA (Active Motif)) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We were able to map R-loop dynamics genome-wide in HCT116 cells in biological triplicates. MapR signal was significantly increased around the transcription start site in the absence of DDX41

2021-11-17 | GSE183081 | GEO

Genome wide mapping of DDX41 chromatin binding using a targeted nuclease approach in U2OS cells after expression

Project description:Purpose: Investigation of DDX41 chromatin binding sites in U2OS cells Method: Expression of DDX41-GFP was induced for 48 hours before crosslinking using 1µg/ml docycycline. Control cells were not induced with doxycline. Cells were mildly crosslinked using 1% FA for 2 min at RT. Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2).1*10^6 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 1 ml Wash Buffer. Cells were permeabilized with 0.05% digitonin in Wash buffer for 4 min at RT. 1 µg Nanobody-GFP-MNase (in-house protein production, PMID:25362362) binding was performed at 4°C for 30 min in 0.05% digitonin-Wash buffer. After 2x washing, the MNase was activated by adding 3mM CaCl2 on ice for 30 min. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, )) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We succesfully mapped DDX41 binding sites on cromatin in U2OS cells. DDX41 preferentially binds in the promoter region of genes.

2021-11-17 | GSE183083 | GEO

MapR in U2OS and HCT116 cells and RNA-seq in U2OS

Project description:MapR in U2OS and HCT116 cells and RNA-seq in U2OS

| PRJNA706388 | ENA

A technique to detect native R-loops genome-wide without immunoprecipitation

Project description:We adapted the nuclease-targeting approach CUT&RUN (Skene & Henikoff, eLife 2017) to map R-loops in vivo. We show that this approach accurately identifies R-loops that are sensitive to both Actinomycin D and RNase H and largely overlap with previously identified regions containing R-loops. We also show that MapR can identify R-loops genome-wide from much smaller input material than existing techniques.

2019-10-18 | GSE120637 | GEO

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instabilityR-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

Project description:Transcription can pose a threat to genomic stability through the formation of R-loops that obstruct the progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RDProx to identify proteins that regulate R-loops in human cells. RDProx relies on the expression of the hybrid-binding domain (HBD) of Ribonuclease H1 (RNaseH1) fused to the ascorbate peroxidase (APEX2), which permits mapping of the R-loop proximal proteome using quantitative mass spectrometry. We associated R-loop regulation with different cellular proteins and identified a role of the tumor suppressor DEAD box protein 41 (DDX41) in opposing R-loop-dependent genomic instability. Depletion of DDX41 resulted in replication stress, double strand breaks and increased inflammatory signaling. Furthermore, DDX41 opposes accumulation of R-loops at gene promoters and its loss leads to upregulated expression of TGFβ and NOTCH signaling genes. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia (AML) in adulthood. We propose that accumulation of co-transcriptional R-loops, associated gene expression changes and inflammatory response contribute to the development of familial AML with mutated DDX41.

2021-11-25 | PXD024517 | Pride

Genome wide mapping of DDX41 chromatin binding using a targeted nuclease approach in U2OS cells after expression

Project description:Genome wide mapping of DDX41 chromatin binding using a targeted nuclease approach in U2OS cells after expression

| PRJNA759136 | ENA

RNA-seq after DDX41 knockdown in U2OS cells

Project description:RNA-seq after DDX41 knockdown in U2OS cells

| PRJNA706394 | ENA

RNA-seq after DDX41 knockdown in U2OS cells

Project description:Purpose: Investigating transcriptome changes after depletion of DDX41. Methods: mRNA profiles after DDX41 knockdown compared to control knockdown were generated in 5 biological replicates. Samples were mapped using STAR (v2.7) against hg38 with the Gencode annotation. Reads per gene were counted using featureCounts (v.1.6). The differential expression analysis was performed using Bioconductor (v2.46)/DESeq2 (v1.26). Genes were deemed sign. diff. regulated with an FDR below 1% Results: We were able to map 32,705 transcripts. Reactome pathway analysis revealed members of the NOTCH and TGFß family to be significantly upregulated when comparing a control knockdown to DDX41 knockdown. In addition, genes in chromatin organization were upregulated.

2021-05-04 | GSE168171 | GEO

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