Loss of matrix integrity underlies age dependent impairment of epidermal stem cell heterogeneity
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF
ORGANISM(S): Cricetulus Griseus (ncbitaxon:10029) Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Hiromi Yanagisawa, M.D. Ph.D.
PROVIDER: MSV000088142 | MassIVE |
REPOSITORIES: MassIVE
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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.
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Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4°C, the cell pellets were resuspended in 100 μl ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4°C). 100 μl of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4°C overnight with 6 μg/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 μl pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 μl ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 μl H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.
2016-07-24 | E-GEOD-77854 | biostudies-arrayexpress
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.
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Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other
2005-04-07 | GSE2408 | GEO
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Project description:We used a 3-step protocol that separates lipid-associated proteins based on their buoyant density, size and charge. All steps were conducted either on ice or at 4C. Step 1 involved a sucrose density gradient centrifugation in which ca. 100 postnatal day 4 rat brains were homogenized in buffer A (5 mM MES, pH 7.0, 0.3 M sucrose, 1 mM EDTA) containing protease inhibitor cocktail. The homogenate was centrifuged at 1,000xg for 15 min. Post-nuclear supernatant (PNS) was lysed with 10 volumes of 5 mM MES pH 7.0, 1 mM EDTA and incubated for 30 min at 4C. The lysed PNS was then centrifuged at 100,000 x g for 1 h. The pellet was resuspended in buffer A and loaded on top of a discontinuous sucrose gradient of 0.8, 1.2 and 2.0 M. The gradient was spun for 3 h at 270,000 x g. The material between 0.3 and 0.8 M sucrose was removed, diluted with 5 mM MES, pH 7.0, 1 mM EDTA to a final sucrose concentration of 0.3 M, and centrifuged at 100,000 x g for 1 h. The pellet was further resuspended in a final volume of 2 mL of PBS with protease inhibitors. In step 2, this material was applied to a gel filtration column packed with Sephacryl S-1000 Superfine matrix. The dimensions of the column were 1.5 cm wide and approximately 1.5 m tall. Material flowed through the column by gravity with elution by 1x phosphate buffered saline (PBS, pH 7.4). The flow rate was ca. 0.5 mL/min, and a BioRad Model 2110 fraction collector was used to collect 4 mL per fraction. For the final step 3 of the protocol, the four Sephacryl S-1000 fractions most highly enriched in Piccolo and Bassoon as determined by WB were pooled and applied to a column packed with Q Sepharose Fast Flow. The dimensions of this column were diameter of 2.5 cm, height of approximately 12 cm with a bed volume of 60 mL packed Q Sepharose, and the flow rate using the 2110 fraction collector was 3 mL/min and 10 mL fractions were collected. The final elution profile (all buffers in 20 mM Tris pH 7.0) was 200 mL of 150 mM NaCl to wash, then stepwise elution of 120 mL buffer with the following NaCl concentrations: 210 mM, 290 mM, 350 mM, and 1000 mM. About 40 mL of the 210 mM, 290 mM (Piccolo and Bassoon enriched), and 350 mM NaCl fractions were concentrated to about 1 mL using Amicon Centricon Plus-20 30 kDa cutoff concentrators. The final recovery was 7.5 ug in the 210 mM fraction, and 16 ug in each of the 290 mM and 350 mM fractions. The fractions were precipitated with tricholoroacetic acid, and the protein pellet was stored at -20C until further use.
The second approach to identify proteins associating with Piccolo and Bassoon was an anti-Piccolo IP. The starting material was 10 mg of the fraction between 0.3 and 0.8 M sucrose after density centrifugation (step 1 above). This material was diluted to 0.3 M sucrose with 5 mM MES, pH 7.0 and centrifuged at 100,000 x g for 1 h. The pellet was resuspended and incubated for 1 h with 4 mL of solubilization buffer (5 mM MES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail). Lysate was cleared by centrifugation at 100,000 x g for 1 h. One mL of supernatant was incubated with 2.5 ug anti-Piccolo rabbit antibody or 2.5 ug rabbit IgG for 4 h. A 100 uL Protein A agarose slurry (Roche) was then added and further incubated overnight. Material was transferred to a small column and washed with 20 mL solubilization buffer. Proteins were eluted twice with 200 uL 50 mM Tris, pH 6.5, 100 mM DTT, 2% SDS. This material was then extracted with 4 volumes methanol and 1-volume chloroform. This mixture was combined with water, centrifuged for 90 sec at 16,000 x g. The aqueous phase was removed and extracted again with 3 volumes of methanol. This precipitated protein was collected by centrifugation for 90 sec at 16,000 x g, methanol removed, and the speed-vacuum was used to dry the protein pellet.
The 210 mM, 290 mM, and 350 mM NaCl fractions from the Mono-Q column protocol and the eluates (both IgG alone and anti-Piccolo IPs) from two separate IP experiments were subjected to Multi-dimensional Protein Identification technology.
2016-07-11 | MSV000079897 | MassIVE
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