Genome wide mapping of DDX41 chromatin binding using a targeted nuclease approach in U2OS cells after expression
ABSTRACT: 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. Overall design: U2OS cells that dox-inducible express GFP-tagged DDX41 protein were either induced or not with 1 µg/ml doxycycline for 48h. Control cells did not express GFP-tagged DDX41. Cells were mildly crosslinked. Nanobody-GFP-Mnase was use to perform CUT&RUN.
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 Overall design: R-loop mapping using MapR after 48h knockdown with a control siRNA or a DDX41 siRNA
Project description:0.3g of flowers were ground in liquid nitrogen. The powder was extracted for 30 min [H2] in 1.5 ml of lysis buffer (50mM Tris HCl pH 8.0, 50mM NaCl, 1% Triton x-100 and 1 x cOmplete™ EDTA-free protease inhibitor (Roche)). After removal of cell debris by centrifugation (2 times 10 min, 16000 x g, 4 °C) the cleared supernatants were incubated for 30 min with GFP or FLAG antibodies coupled to magnetic microbeads (μMACS GFP and DYKDDDDK isolation Kits, Miltenyi). Beads were loaded on magnetized MACS separation columns equilibrated with lysis buffer and washed 4 times with 300 µl washing buffer (50mM Tris HCL pH 7.5, 25 or 50 mM NaCl, 0,1 % Triton). Samples were eluted in 50 µl of pre-warmed elution buffer (Milteny). Control IPS were performed in Col-O using either GFP or FLAG antibodies.
Project description:Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are underexplored. Here we report that a novel somite-derived pro-hematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch upregulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide ChIP-seq, RNA-seq, and ATAC-seq indicate that Srebp2 trans-regulates Notch pathway genes required for hematopoiesis. Our studies outline a novel AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease. Overall design: The ATAC-Seq was performed using 50,000 c-Kit+CD144+ CD45.2- cells isolated from AGM region of E11.5 wild-type B6 mouse embryos. The Tn5 transposome was purified and assembled following a published protocol (46). The permeabilization was performed with 50 µl cold ATAC-RSB buffer (0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) and the transposition was performed with 50 µl transposition mix (10µl 5× HEPES DMF Buffer, 3 µl 5 µM Tn5 transposome, 37 µl PBS, 0.5 µl 10% Tween-20, and 0.5 µl 1% digitonin) and incubated at 37° for 30 min. After transposition, the cleaned-up DNA fragments were pre-amplified for 5 cycles using NEB Q5 master mix. Each reaction contains 2.5 µl of 25 µM i5 primer, 2.5 µl of 25 µM i7 primer, 25 µl 2× NEB Q5 master mix, and 20 µl cleaned up samples. PCR settings were 5 min at 72°C, 30 sec at 98°C, and followed by additional 5 cycles (98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min). After pre-amplification, 1 µl of the pre-amplified mixture was used to run a 10 µl qPCR test to determine the optimal amplification cycle. The final amplified DNA library was purified using Qiagen DNA purification kit and sequenced on NextSeq 500 with SE75 strategy. This submission represents the ATA-Seq component of study.
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. Overall design: ATACseq in T. brucei wild-type, H3.V-/-, H4.V-/- and H3.V-/- H4V-/- cells. Each performed in duplicates with 10 and 20 million cells, respectively. A gDNA control was included to check for biases in accessiblity.
Project description:The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1?µL eluent in 9?µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2-2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.
Project description:A selective and sensitive gradient HPLC-UV method for quantification of sotalol hydrochloride and potassium sorbate in five types of oral liquid preparations was developed and fully validated. The separation of an active substance sotalol hydrochloride, potassium sorbate (antimicrobial agent), and other substances (for taste and smell correction, etc.) was performed using an Ascentis Express C18 (100?×?4.6?mm, particles 2.7??m) solid core HPLC column. Linear gradient elution mode with a flow rate of 1.3?mL?min(-1) was used, and the injection volume was 5?µL. The UV/Vis absorbance detector was set to a wavelength of 237?nm, and the column oven was conditioned at 25°C. A sodium dihydrogen phosphate dihydrate solution (pH 2.5; 17.7?mM) was used as the mobile phase buffer. The total analysis time was 4.5?min (+2.5?min for reequilibration). The method was successfully employed in a stability evaluation of the developed formulations, which are now already being used in the therapy of arrhythmias in pediatric patients; the method is also suitable for general quality control, that is, not only just for extemporaneous preparations containing the mentioned substances.
Project description:Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2. Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using Lipofectamine™ RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturer’s recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x Complete® protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a Bioruptor™ UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 ⁰C with rotation. Antibodies were immunoprecipitated with 60 µl protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 ⁰C for 2 hours. Next, proteins and RNA were digested by adding 33 µl of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 ⁰C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using Qubit™ (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.
Project description:It is desirable to develop a fast method for quantification of melphalan due to its instability. Here we report a method for quantification of melphalan (MPL) in human plasma using a UPLC-PDA system. Briefly, 50 µL plasma sample was mixed with 25 µL internal standard (2500 ng/mL acetylmelphalan in methanol) and 25 µL 20% trichloroacetic acid, and centrifuged at 21,000 g (15,000 rpm) at 4 °C for 3 min. The supernatant (5 µL) was injected onto an Acquity™ BEH C18 LC column (2.1?×?50 mm, 1.7 µm) and eluted with 25 mM NH4AC (pH 4.7)-acetonitrile in a gradient mode at a flow rate of 0.6 mL/min. The column kept at 40?±?5 °C and the autosampler kept at 4?±?5 °C. The detector set at 261 nm, and sampling rate was 40points/sec. The retention times were typically 2.11 min for melphalan and 2.38 min for the internal standard. Total run time is 4 min per sample. Calibration range was 100-40,000 ng/mL. The lower limit of quantification was 100 ng/mL. The method was validated based on the FDA guidelines, and applied to a clinical pharmacokinetic study in pediatric patients.
Project description:Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. U2OS cells were grown in DMEM supplemented with 10% FCS, 100 U/l penicillin, and 100 μg/l streptomycin at 37 °C in 10% CO2 and passaged at ~80% confluence. U2OS cells expressing LAP1-tagged MRFAP1 were grown in the same medium but with the addition of 150 μg/ml hygromycin B and 15 μg/ml blasticidine HCl. The cells for each condition were harvested separately via trypsinization, washed in PBS, and lysed in IP buffer (1% Nonidet P-40, 50 mm Tris-HCl, pH 7.4, 10% glycerol, 150 mm NaCl, complete protease inhibitor mixture (Roche Applied Science), PhosStop, 50 mm N-ethylmaleimide). The lysates were sonicated for 10 s at 10% power (three times in total) and then centrifuged for 10 min at 17,000g at 4 °C. Equal protein amounts of each sample were then combined with GFP-trap agarose beads from ChromoTek (Martinsried, Germany) that had been washed once in IP buffer (40 μl of 50% GFP-trap bead slurry per IP) and incubated for 2 h at 4 °C with rotation. The beads were then washed three times with IP buffer via centrifugation at 2,000g for 2 min at 4 °C.
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. Overall design: Examination of pSer2-Pol II profile