Cleavage Under Targets and Tagmentation（CUT&Tag）analysis of ovarian cancer cells treated by Paeonol
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE194318 | GEO | 2025/01/18
REPOSITORIES: GEO
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Project description:Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modelled this leukemic evolution through stepwise gene editing of GATA1s, SMC3+/- and MPLW515K providing 20 different trisomy or disomy 21 iPSC clones. Cut&Tag against GATA1 was performed on CD41+CD42+ megakaryocytes obtained after 18 days of differentiation of the IPSC clones. 500,000 CD41+CD42+ MK sorted cells were used to analyze GATA1 and GATA1s chromatin occupancy using the CUT&Tag-IT Assay Kit (Active Motif) according to the manufacturer recommendations. Briefly, cells were bound to Concanavalin A-Coated Beads and incubated with primary anti-GATA1 antibody in buffer with Protease Inhibitor Cocktail and 5% digitonine overnight at 4°C under rotation. The Guinea Pig anti-rabbit secondary antibody was incubated in Dig-Wash buffer for 1 hour at RT under rotation. After 3 washes, the CUT&Tag-IT™ Assembled pA-Tn5 Transposomes (1:100) were added for 1 hour at RT under rotation and tagmentation was performed during 1 hour at 37°C. DNA was purified and libraries were generated by PCR. The final libraries were purified, pooled together in equal concentrations and subjected to paired-end sequencing (100 cycles: 2x50) in Novaseq-6000 sequencer (Illumina) at Gustave Roussy.
2022-09-30 | E-MTAB-11032 | biostudies-arrayexpress
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
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
Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.
2021-09-05 | GSE152862 | GEO
Project description:Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed “TRACE-seq”) are comparable to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis; at the meantime, TRACE-seq enables a one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.
2020-07-29 | GSE143422 | GEO
Project description:Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from sub-picogram amounts of cDNA. Comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, as naked Tn5 can be annealed to any oligonucleotide of choice, for example molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enables innovation in sequencing-based applications.
2014-07-14 | GSE58652 | GEO
Project description:Goal of experiment: Identification of differentially expressed immune genes from male and female BWF1 lupus-prone mice. (Female incidence is higher than male--attempting to find sex hormone regulated genes that may contribute to this difference). Whole spleen was taken from pre-lupus (4 months old) BWF1 (females are lupus-prone) male and female mice. Preparation of cDNA. Double-stranded cDNA was synthesized from purified RNA. The first strand was synthesized by incubating 5 μg of RNA with 100 pg/ml T7-(dT)24 primer (HPLC purified DNA primer sequence: 5’-GGCCAGTGAATTGTAATACG ACTCACTATAGGGAGGCGG-(dT)24 -3’ Genset Corp, San Diego, CA) at 70°C for 10 minutes. Samples were incubated for 1 hour at 42°C with the following mix: 1X first strand buffer, 10 mM dithiothreitol, 500 μM each dNTP, 200 U SuperScript II in diethylpyrocarbonate (DEPC)-treated water up to 20 μl. Second strand synthesis was performed by incubating the first strand with the following mix for 2 hours at 16°C: 1X second strand reaction buffer, 200 μM dntps, 10 U E. coli DNA ligase, 40 U E coli DNA Polymerase I, 2 U of E. coli RNase H up to 150 μl with DEPC-treated water (all reagents were contained in SuperScript Choice System for cDNA Synthesis, Invitrogen). A phenol/chloroform extraction was performed on the ds-cDNA preparation before biotin-labeled cRNA was generated. Synthesis and fragmentation of biotin-labeled cRNA (in vitro transcription). The ENZO BioArrayTM HighYieldTM RNA Transcript Labeling Kit (T7) (Enzo diagnostics, Inc., Farmingdale, NY) was used to produce large amounts of hybridizable biotin-labeled RNA targets by in vitro transcription from the ds-cDNA. The following mix was incubated at 37°C for 5 hours: 1 μg of ds-cDNA, 1X HY reaction buffer, 1X biotin labeled ribonucleotides, 1X dithiothreitol, 1X T7 RNA Polymerase. Biotin-labeled cRNA was run over RNeasy spin columns (Qiagen), quantified, and run on an agarose gel to visualize the size distribution of labeled transcripts. Twenty micrograms of cRNA was incubated with 1X fragmentation buffer for 35 minutes at 94°C. (5X fragmentation buffer: 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc). After fragmentation, the samples were stored at -20°C until the hybridization was performed. Sample hybridization. Oligonucleotide microarrays (MGU74v2 A, B, and C GeneChip probe arrays; Affymetrix) were hybridized with labeled cRNA derived from spleens from individual mice. For each array,15 μg of fragmented cRNA was mixed with a hybridization cocktail consisting of 1X hybridization buffer (2X hybridization buffer: 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween), 0.5 mg/ml acetylated BSA (Invitrogen), 0.1 mg/ml herring sperm DNA (Promega), and water (BioWhittaker) up to 300 μl). Biotin labeled cRNA transcripts of the E. coli and P1 bacteriophage genes, BioB, bioC, bioD, and cre (GeneChip Eukaryotic Hybridization control kit, Affymetrix) were spiked into each hybridization mix at 1.5, 5, 25, and 100 pM to evaluate sample hybridization efficiency for each array. The hybridization cocktail was heated to 99°C and then 45°C for 5 minutes each before it was centrifuged to remove any insoluble material. The array was equilibrated to room temperature, moistened with 1X hybridization buffer, and incubated for 10 minutes at 45°C with rotation. After incubation, the buffer solution was removed from the array. The array was filled with 300 μl of the hybridization cocktail, placed in a rotisserie box in a 45°C oven, and incubated for 16 hours while rotating at 60 rpm. Washing and staining of array. The hybridization cocktail was removed and the GeneChip Fluidics Station 400 (Affymetrix) with Microarray Suite software (Affymetrix) was used to wash and stain the probe arrays with the following protocol: 10 cycles of 2 mixes/cycle with wash buffer A at 25°C, 4 cycles of 15 mixes/cycle with wash buffer B at 50°C, 30 minute incubation with staining solution at 25°C, 10 cycles of 4 mixes/cycle with wash buffer A at 25°C. Wash buffer A -- non-stringent wash buffer (6X sodium chloride sodium phosphate + ethylenediaminetetraacetic acid (SSPE), 0.01% Tween-20). (20X SSPE: 3 M NaCl, 0.2 M NaH2PO4, 0.02 EDTA) (BioWhittaker). Wash buffer B – stringent wash buffer (100mM MES, 0.1 M [Na+], 0.1% Tween 20). Staining solution (1X 2-(N-Morpholino)ethanesulfonic Acid (MES) stain buffer, 2 mg/ml acetylated BSA, 10 μg/ml Streptavidin Phycoerythrin (SAPE), and water up to 600 μl). (12X MES stain buffer: 1.22 M MES, 0.89 M [Na+]). Analysis. After staining, the probe arrays were scanned using the GeneChip 3000 Scanner (Affymetrix) with Microarray Suite software (Affymetrix). Technical and assay variation between arrays was corrected for by multiplying or dividing the overall intensity of each array by a scaling factor so that the overall intensity of each array was equivalent to facilitate comparison analysis. Keywords = BWF1 total spleen Keywords: repeat sample
2005-08-23 | GSE2336 | GEO
Project description:To determine the biological function of ATF4 in the modulation of downstream target genes, we performed Tagmentation (CUT&Tag) assay in HCT 116 (Human colorectal cancer) cells
2022-11-29 | GSE218559 | GEO
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
Project description:We identified a SNP rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple ChIP-seq experiments, and its strong-binding allele increased transactivation, and accorded higher mRNA levels in cell lines and human brain. To confirm the allele-specific binding, we conducted ChIP tagmentaion sequencing experiment in human lymphoblastoid cell line GM12763 which is heterozygous for rs242561. NRF2 ChIP DNA was isolated from GM12763 cells treated with Sulforaphane (SFN) in triplicates. The region containing rs242561 was amplified using primers, Fwd 5â??-AGCCTTCCCTGTCCTTGATT-3â??, Rev 5â??-GGACCGAGCTTCCAGTCTAA-3â??, and tagmentated using Tn5-based transposition for library construction. Libraries were sequenced on Illumina MiSeq. NRF2 ChIP tagmentation sequencing of a 241bp intron region in MAPT gene using GM12763 treated with sulforaphane
2016-04-14 | E-GEOD-79569 | biostudies-arrayexpress