Genome wide mapping of DDX41 chromatin binding using a targeted nuclease approach in U2OS cells after expression
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE183083 | GEO | 2021/11/17
REPOSITORIES: GEO
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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:Approximately 3 × 105 NPCs sorted for Sox2-eGFP were mixed with 3 × 104 Drosophila S2 cells per reaction. We performed CUT&RUN using the protocol developed by the Henikoff lab (Skene and Henikoff, 2017). In brief, Bio-Mag®Plus Concanavalin-A (Con A) coated beads (Bangs Laboratories BP531) were washed and activated with Binding buffer. Nuclei of NPCs with S2 spike-in were gently prepared (see Subcellular Protein Extraction section). Activated Con A beads and nuclei were then mixed and rotated for 5 min at room temperature. They were then blocked with Digitonin block buffer for 5 min at room temperature. 0.5-1 μg of primary antibody with 0.25 μg Spike-in antibody (Active Motif 61686) diluted in Digitonin block buffer was added to the bead-nuclei mixture and incubated for 3 hours (histone marks) or O/N (the rest) at 4 °C. Beads were washed three times with Digitonin block buffer and incubated with pA-MNase for 1 hour at 4 °C. Beads were washed three times with Wash buffer and incubated in Wash buffer for 10 min on ice. The pA-MNase was activated by incubating the beads with 2 mM CaCl2 for 25 min on ice and quenched by adding the Stop buffer. DNA was released from the beads by incubating them for 30 min at 37 °C and collected by centrifugation at 16,000 x g for 5 min at 4 °C. DNA was then isolated by using a phenol/chloroform extraction method.
2023-07-12 | GSE212444 | GEO
Project description:The aim of the project was to purify Xenopus replisome from replicationg chromatin assembled in Xenopus laevis egg extract. To this end recombinant Cdc45-TEV-His10-FLAG5 was expressed in bacteria and purified. 4 ml of Xenopus laevis egg extract was activated into interphase and supplemented with 10 ng/µl of demembranated sperm DNA, 70 nM recombinant Cdc45, 40 µM aphidicolin, 5 mM caffeine and incubated at 23°C for 60 min. Chromatin was isolated in ANIB100 buffer (50 mM HEPES pH 7.6, 100 mM KOAc, 10 mM MgOAc, 2.5 mM Mg-ATP, 0.5 mM spermidine, 0.3 mM spermine, 1 µg/ml of each aprotinin, leupeptin and pepstatin, 25 mM β-glycerophosphate, 0.1 mM Na3VO4 and 10 mM 2-chloroacetamide) as described previously (Gillespie, Gambus et al. 2012). Chromatin pellets re-suspended in ANIB100 containing 20% sucrose. Protein complexes were released from chromatin by digestion with 4 U/µl benzonase nuclease (E1014-25KU, Sigma) and sonicated for 5 min using a sonicator with settings: 30 sec on, 30 sec off, low power (Bioruptor Diagenode UCD-200). Immunoprecipitation was performed using 100 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich). Before elution the sample was washed four times with 1 ml of ANIB100 20% sucrose, ANIB100 20% sucrose 0.1% Triton X-100, ANIB100 and elution buffer (25 mM HEPES pH 7.5, 100 mM KAc, 5 mM MgAc, 1 mM ATP and 0.02% NP-40), respectively. The sample was eluted adding 250 µM 3xFLAG peptide (Stratech) to 200 µl of elution buffer and a small proportion of it analysed by mass spectrometry.
2023-10-27 | PXD044422 | Pride
Project description:Ten 15 cm2 plates of HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were lysed with TAP lysis buffer (10% (v/v) glycerol, 50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM EDTA, 0.1% (v/v) NP-40, 10 mM NaF, 0.25 mM Na3VO4, 50 mM β-glyerolphosphate, 2 mM DTT, and 1X protease inhibitor cocktail (Sigma)) and cleared supernatants were bound to IgG-Sepharose beads (GE Amersham) and then washed in lysis buffer and TEV buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM DTT, and 1X protease inhibitor cocktail). Protein complexes were cleaved off the beads by TEV protease (Sigma) and incubated with calmodulin-Sepharose beads (GE Amersham) in calmodulin binding buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 0.1% (v/v) NP-40, 6 mM CaCl2, 10 mM 2-mercaptoethanol) and then washed in calmodulin rinse buffer (50 mM Ammonium bicarbonate pH 8.0, 75 mM NaCl, 1 mM Mg Acetate, 1 mM Imidazole, 2 mM CaCl2) before eluted with SDS sample buffer, subjected to SDS-PAGE, and stained with GelCode Blue stain (Thermo Fisher Scientific) before protein identification by LC-MS/MS.
2020-09-01 | PXD020231 | Pride
Project description:Purpose: this study is to analyze the chromatin landscape of H3R2mes in Plasmodium falciparum. Methods: In this study, H3R2me2s enrichment in the chromatin of wildtype parasite at schizont stage was analyzed by Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with next generation sequencing (CUT&Tag-seq). Synchronized WT 3D7 at 40–46 hpi schizonts were harvested and fixed with 1% formaldehyde for 1 min at room temperature, followed by lysis with 0.06% saponin. The parasite pellets were suspended in 100 μl nuclear extraction buffer (20 mM HEPES–KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1x EDTA-free Protease Inhibitor cocktail). Ten μl of activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei) and incubated for 10 min. The bead-bound nuclei were resuspended in 50 μl Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail, 0.01% digitonin, 2 mM EDTA) containing 0.5 μg of rabbit anti-H3R2me2s (Epigentek #A-3705-050) as the primary antibody or rabbit IgG (Cell Signaling Technology #2729S) serving as a control. The primary antibody was then removed, and nuclei were then incubated with 0.5 μg of the secondary antibodies in 50 μl of Digitonin150 buffer (Antibody150, no EDTA) at room temperature for 30 min. The secondary antibodies were mouse anti-rabbit IgG (Invitrogen #31213). The nuclei were washed thrice with 200 μl Digitonin150 Buffer for 5 min to remove unbound antibodies and finally resuspended in 50 μl Digitonin300 Buffer (Digitonin150 but with 300 mM NaCl). Then, 2.5 μl of CUTANA pAG-Tn5 (EpiCypher #15-1017) were added to each reaction at room temperature for 1 h. The nuclei were washed thrice for 5 min in 200 μl Digitonin300 Buffer to remove the unbound pA-Tn5. Next, the nuclei were resuspended in 50 μl Tagmentation buffer (10 mM MgCl2 in Digitonin300) and incubated at 37 °C for 1 h. The beads were washed with 50 µl TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA), mixed with 5 µL SDS release buffer (10 mM TAPS pH 8.5; 0.1% SDS) to quench tagmentation, and then incubated for 1 hr at 58ºC to release tagmented chromatin into solution. Finally, 15 µl of 0.67% Triton-X was added to each reaction to quench SDS. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by incubating with 1.3 × Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and next-generation sequencing was performed on the NextSeq 550 platform. The reads of CUT&Tag-seq from three biological replicates were mapped to the P. falciparum genome (PlasmoDB Release 39.0) by using BWA (Li and Durbin 2009) after FastQC quality control. The peak calling was finished by MACS2 version 2.2.7.1 with parameters ‘callpeak -f BAMPE’ and a q-value cutoff of 0.05. Results: The three replicates of CUT&Tag-seq using nuclei from the schizont stage showed high reproducibility, with correlation coefficients of ~0.8. Peak calling using criteria of presence in at least two of three replicates with at least 50% overlap of the peaks identified 1629 H3R2me2s signals, distributed among the 14 chromosomes. Among these peaks, 57% (927) are localized at 5’ UTR regions of 791 genes, 35% (577) at coding regions of 470 genes, and 8% (125) at 3’ UTR regions of 115 genes. The peaks are ~17.44 bp wide and ~1265 bp from the ATG sites. Comparing the transcriptome data showed genes with H3R2me2s enrichment in the 5’ UTRs showed significantly higher expression than other genes in the genome, suggesting that H3R2me2s is associated with gene activation. In addition, H3R2me2s peaks were highly co-localized with the active mark H3K9Ac, H2A.Z, and H3K4me3, further implying H3R2me2s as a euchromatin mark. Gene ontology (GO) enrichment analysis revealed a multitude of cellular pathways, including merozoite invasion, transcription, translation, stress response, cell cycle, schizogony, cell adhesion, exit from RBC, vesicle transport, signal transduction, DNA repair, and telomere organization, which are potentially regulated by this euchromatic mark. Conclusions: Collectively, H3R2me2s is a active chromatin mark and regulates invasion and other important processes in human malaria parasite.
2023-06-28 | GSE214535 | GEO
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:The goal of this study is to analyse the open chromatin regions via ATACseq in zebrafish embryonic endothelial cells at 26-27hpf using Tg(Kdrl:GFP) transgenic zebrafish embryos. 40000-50000 cells were FAC-sorted directly into 100 µl of 1x HBSS/10 mM HEPES/0.25% BSA buffer for Tagmentation (as described by Buenrostro et al. (2013), Nat. Methods 10, 1213-1218), but with 1.5 µl Tn5 transposase (Illumina) in a 50 µl reaction volume. DNA was purified using the QIAquick PCR purification kit (Qiagen). Fragments were amplified for 16 cycles.
2020-01-16 | GSE143763 | GEO
Project description:3x107 cells were harvested and washed in 30 ml cold 1x TDB. The pellet was resuspended in 300 µl permeabilization buffer with protease inhibitors, 3 µl of 4 mM digitonin was added and incubated for 5 minutes at RT. The cells were pelleted, resuspended in 600 µl isotonic buffer with protease inhibitors and split in two samples, containing 1x107 and 2x107 cells, respectively. The transposition reaction was performed by adding 50 µl of transposition mix to the pellet (25 µl TD (2x reaction buffer from Nextera kit), 25 µl TDE1 (Tn5 transposase from Nextera kit), 22.5 µl nuclease-free water) and incubation for 30 minutes at 37 °C. For the gDNA control, 200 ng of gDNA was treated in the same manner. The DNA samples were purified using Qiagen MinElute PCR Purification Kit and eluted in 10 µl EB (10 mM Tris-HCl, pH 8). The transposed DNA fragments were amplified using the NEBNext® High-Fidelity 2X PCR Master Mix (M0541) supplied with 2.5 µl of 25 µM barcoded primers and amplification for 13 cycles. The libraries were purified using AMPure XP beads (Beckman Coulter) following the manufacturer’s instructions. The library fragment sizes between 150 and 1000 bp were purified from a 6% polyacrylamide gel. Paired-end 76 bp sequencing was carried out using the Illumina NextSeq system with a mid-output NextSeq 500/550 kit according to the manufacturer’s instructions.
2018-08-24 | GSE118902 | GEO
Project description:The aim of this project was to determine which viral and host proteins bind to purified recombinant human GTPase Rab11a from lysates of HEK-293T cells infected with influenza A/WSN/33 (H1N1) virus. To this aim we used a GST pull-down assay. Approximately 8∙106 HEK-293T cells were infected with influenza A/WSN/33 (H1N1) virus at a multiplicity of infection of 5 (MOI=5) or mock infected. Twelve hours (h) post-infection cells were harvested by centrifugation at 1,000 rpm for 5 min and lysed for 1 h in lysis buffer [50 mM HEPES-NaOH pH 7.5, 200 mM NaCl, 0.5 % (v/v) IGEPAL CA-630 (Sigma-Aldrich), 1 mM β-mercaptoethanol, 1x PMSF, 1x protease inhibitor (Roche, complete mini, EDTA-free)] at 4 °C. Supernatants were collected by centrifugation at 13,000 rpm for 5 min at 4 °C. Approximately 0.2 mg of purified GST-tagged Rab11CA (constitutively active Rab11a with a Q70L substitution) or GST tag was bound to pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) (100 µl slurry per 1 ml sample volume) at 4 °C for 3 h in binding buffer [50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 10 % (v/v) glycerol, 8 mM MgCl2, 10 µM GTP-γ-S (Abcam)]. GST-Rab11CA or GST tag bound beads were incubated with virus infected or mock infected cell lysates for 1 h at 4 °C followed by five washes in binding buffer. Rab11CA and bound proteins were released in the same buffer supplemented with 1 mM DTT and 0.1 mg human rhinovirus (HRV) 3C protease to cleave off Rab11CA from the GST tag. After 1 h incubation at 4 °C released proteins were cleared from the beads by centrifugation at 1,000 rpm for 5 min at 4 °C. GST pull-down protein fractions were used for analysis by mass spectrometry.
2022-01-17 | PXD030058 | Pride