Mass spectrometry was used to identify proteins that bind to S3B
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Qiong Luo
PROVIDER: PXD055774 | iProX | Thu Sep 12 00:00:00 BST 2024
REPOSITORIES: iProX
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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
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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.
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2021-09-05 | GSE152862 | 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
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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