MS analysis of STING linear ubiquitinated sites
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Lingqiang Zhang
PROVIDER: PXD046415 | iProX | Thu Oct 26 00:00:00 BST 2023
REPOSITORIES: iProX
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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: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).
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Project description:The cells were cotransfected with epitope-tagged expression plasmids or empty vector(EV) . Forty-eight hours after transfection, the cells were harvested with 500 μl of lysis buffer (20 mM Tris-HCl, pH=8.0; 100 mM NaCl; 0.5% NP-40; and 1 mM EDTA) supplemented with protease inhibitor cocktail (#04693132001, Roche) and PMSF (#PB0425). The cell lysate was rotated at 4°C for 30 min. After centrifugation at 13 000 × g for 10 min, the supernatants were used for immunoprecipitation (IP). The cell extracts were incubated with anti-Flag antibody (F1804 Sigma) overnight at 4°C, followed by incubation with lysis buffer and washing with 50 μL of protein G agarose resin (Yeasen, 36405ES08) for 3 h. The incubated protein G was washed with lysis buffer at 4°C for 10 min. The cell extracts (50 μL of input) and IP samples were resuspended in 80 μL of SDS buffer, followed by pipetting up and down several times to mix the samples.For mass spectrometry analysis of heterochromatin-associated proteins and murine Nat10-associated proteins, the immunoprecipitate was subjected to 10% Acr-Bis SDS‒PAGE and separated approximately 2 cm from the well. The gel was cut and used for mass spectrometry analysis by the PTM BIO Company (Hangzhou, China).
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Project description:gDNA was estracted and digested with cocktail of restrict enzymes MseI, DdeI, AluI, MboI, incubated at 37°C for 6 h. After purified by phenol/chloroform extraction, fragmented DNA was resuspended by TE, and quantified by Qubit 3.0 (Invitrogen). For each sample, 20 μg input DNA was immunoprecipitated overnight at 4°C in a shaker, with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 μg S9.6 antibody (ATCC, HB-8730). After adding 50 μl Dynabeads Protein G (Invitrogen, 10004D) and incubated for 4 h, the beads with antibody were washed by 1× DRIP binding buffer for 4 times at room temperature, each wash takes 10 min. Added 250 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to beads/antibody complexes, incubated for 40 min in an Eppendorf ThermoMixer at 55°C, 1,000 rpm. Purified by phenol/chloroform extraction, and moved supernatant to a new tube, followed by adding 1/10 volume 3 M NaAc, 1 μl GlycoBlue (Invitrogen) and 1 volume isopropanol, precipitated at -20°C for 3 h and then centrifuged. After washed by 70% ethanol and dried, the DRIPed DNA pellet was resuspended by 75 μl low EDTA TE buffer (10 mM Tris-HCl 8.0, 0.1 mM EDTA). NGS libraries were constructed using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences).
2019-10-15 | GSE134101 | GEO
Project description:We generated a HEK293 cell line expressing ZZ-TEV-FLAG-bait protein by Lipofectamine 3000 transfection. The expression level of bait protein is controlled to endogenous level by addition of 2 ng/mL doxycycline. Note the combined treatment of Torin1 and CQ was applied for STX17. The tandem affinity purification of tagged bait protein was performed in a physiology buffer condition [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Igepal CA-630, 1 mM EDTA, protease inhibitor cocktail (Roche)]. The eluted protein mixtures were subjected to SDS/PAGE and further analyzed by mass spectrometry.
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Project description:Human keratinocytes, HaCaT cells were transfected with the dsRNA-analogue polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich) using Lipofectamine 2000 reagent (Invitrogen) for intracellular delivery. The cells were transfected with 7 ug/ml poly I:C for 1 h or left untreated. The cells were collected in PBS with a cell scraper and washed once with PBS before they were lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The cell lysates were centrifuged 2,264 x g for 15 min at 4°C, divided into smaller fractions and centrifuged again 11,686 x g for 15 min at 4°C . The supernatant was collected and the protein content was measured with Bio-Rad DC protein assay (Bio-Rad). For the samples, 8 mg of protein was used. The proteins were precipitated with 10 % TCA/acetone at -20°C overnight. Samples were centrifuged 10,733 x g for 20 min at 4°C and the supernatant was removed. The precipitation was washed once with acetone and dissolved in urea buffer (8 M urea, 400 mM NH4CO3, 20 mM DL-dithiotheitol, 1 mM EDTA, pH 8.5). The proteins were reduced, alkylated, and enzymatically digested in-solution with lysyl endopeptidase (Wako Chemicals) and trypsin (Promega). The peptides were fractionated by strong cation exchange chromatography. Phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich). The enriched phosphopeptides were analyzed by nanoLC-MS/MS using an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization.23,24 The LC-MS/MS data were submitted through the ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) to an in-house Mascot database search engine version 4.0 (Matrix Science), and to the ProteinPilot algorithm Paragon. The data were searched against the human canonical sequences in the Swiss-Prot database (decoy version 08132012 for the Mascot searches and version 04012013 with 539,829 sequences for the Paragon searches). The Mascot search criteria were: fixed modifications: carbamidomethyl of cysteine, variable modifications: oxidation (M), phospho (ST), phospho (Y), acetylation (K), 1 missed cleavage allowed, enzyme: trypsin. The Paragon search criteria were: modifications: cystein alkylation with iodoacetamide, ID focus: biological modifications, phosphorylation emphasis, identification threshold: 95 % confidence (Unused ProtScore (Conf) > 1.3), Thorough search (ID), enzyme: trypsin.
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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:During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we find that the interplay between these factors depends on their large-scale genomic context. The mostly unmethylated CpG islands have a reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated and their average density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the level of TET1 and 5hmCs goes down. In particular, this has a dramatic effect at variable CTCF sites enriched outside CpG islands, that are occupied by CTCF in ESCs but loose CTCF during differentiation. To rationalize this cell type dependent targeting of CTCF binding in competition with the histone octamer, we have developed a quantitative biophysical model, which allowed predicting differences in CTCF binding due to TET1/5hmC/5mC-dependent nucleosome repositioning. MNase-seq experiments for analysis of mononucleosomes were conducted as described previously (Teif et al. Nature Struct. Mol. Biol., 2012). In addition, a dinucleosome fraction also was gel purified and sequenced. Briefly, embryonic stem cells from 129P2/Ola mice were cultured in ESGRO complete medium (Millipore), harvested and resuspended in low salt buffer (10 mM Hepes, pH 8, 10 mM KCl, 0.5 mM DTT) at 4 M-BM-0C. After disruption of the cells with a douncer the nuclei were collected by centrifugation and washed once with the MNase Buffer (10 mM TrisM-bM-^@M-^SHCl, pH 7.5, 10 mM CaCl2), resuspended in the MNase Buffer and digested with 0.5 units MNase per microliter (Fermentas) and incubation for 6M-bM-^@M-^S11 minutes at 37 M-BM-0C. The MNase digestion was stopped by putting the samples on ice and adding EDTA to a concentration of 10 mM. After digestion with 0.1 M-BM-5g M-BM-5lM-bM-^@M-^S1 RNase A (Fermentas) and removal of protein by phenol and chloroform extraction, the DNA was ethanol precipitated and the resulting DNA pellet was dissolved in H2O. DNA fragments corresponding to mononucleosomes or dinucleosomes were separated on a 2 % agarose gel using an E-Gel electrophoresis system (Life Technologies). The libraries for sequencing were prepared according to the standard Illumina protocol. High-throughput paired-end sequencing of at least 50 bp read length was performed on the Illumina HiSeq 2000 platform at the DKFZ sequencing core facility in Heidelberg, Germany. We obtained about 150 million nucleosome positions per sequencing reaction. ChIP-seq experiments were conducted as described previously (Teif et al. 2012). For each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes, pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 M-BM-5g of either H3K9me3 (Abcam ab8898) or H3K4me3 (Abcam ab8580) antibody over night. After washes with sonication (10 mM TrisM-bM-^@M-^SHCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% NaM-bM-^@M-^Sdeoxycholate), high-salt-buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaM-bM-^@M-^Sdeoxycholate, 0.1% SDS), lithium buffer (20 mM TrisM-bM-^@M-^SHCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% NaM-bM-^@M-^Sdeoxycholate) and 10 mM TrisM-bM-^@M-^SHCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina HiSeq 2000 sequencing platform (single reads of 50 bp length).
2014-05-07 | E-GEOD-56938 | biostudies-arrayexpress