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RNA-seq after DDX41 knockdown in U2OS cells

ABSTRACT: RNA-seq after DDX41 knockdown in U2OS cells

PROVIDER: PRJNA706394 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13848180.fastq.gz	Fastqsanger.gz
	SRR13848181.fastq.gz	Fastqsanger.gz
	SRR13848182.fastq.gz	Fastqsanger.gz
	SRR13848183.fastq.gz	Fastqsanger.gz
	SRR13848184.fastq.gz	Fastqsanger.gz

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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

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 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

Genome-wide maps of double strand breaks after siRNA knockdown of DDX41 in HCT116 cells

Project description:We report the first genome-wide mapping of fragility hotspots after siRNA-mediated knockdown of the DEAD-box helicase DDX41 in HCT116 cells using sBLISS. DDX41 defficient cells display elevated levels of genomic instability. sBLISS analysis revealed that hotspots for genomic instability in the absence of DDX41 are located around the transcription start site of transcribed genes.

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Inherited and somatic defects in DDX41 in myeloid neoplasms

Project description:Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-Box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases lead to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved diagnostic bone marrow or peripheral blood samples.

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Frac-Seq Analysis of ZFC3H1 Knockdown U2OS Cells

Project description:Frac-Seq Analysis of ZFC3H1 Knockdown U2OS Cells

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Mapping perturbations in the cellular proteome of OCIAD1 and OCIAD2 knockdown U2OS cells.

Project description:To gain insight into the function of both OCIAD paralogs, we used untargeted quantitative mass spectrometry to compare the whole-cell proteomes of control U2OS cells, U2OS cells with individual or double OCIAD1/OCIAD2 knockdown, and OCIAD1 knockdown cells in which OCIAD1 expression was reintroduced by lentiviral delivery.

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KLF5 RNA-seq in U2OS cells

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

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

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SRp20 regulates Genome-wide Alternative RNA splicing events in U2OS cells

Project description:To examine the effects of SRp20 on genome-wide RNA splicing in tumor cells, we utilized Exonhit SpliceArray to compare genome-wide changes of RNA splicing and transcription in U2OS cells with or without knockdown of SRp20 by RNAi. U2OS cells were treated by SRp20 siRNA or non-specific siRNA, each performed in triplicate. A human genome-wide splice-array assay was performed by using a service provider ExonHit Therapeutics, Inc (Gaithersburg, MD). Fifty ng of total RNA isolated from U2OS cells with or without knockdown of SRp20 was quality controlled and analyzed by using Affymetrix GeneChip platform to profile the expression of 20,649 genes and splicing events of 19,066 genes.

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