Ena-DATASET-ErasmusMC-hema-22-07-2021-13:40:41:085-493 - samples
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ABSTRACT:
PROVIDER: EGAD00001007910 | EGA |
REPOSITORIES: EGA
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Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)
2024-05-23 | PXD033079 | Pride
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
2019-10-15 | GSE134103 | GEO
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: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: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:Samples were purchased from Clontech as mRNA. Hybridization material was generated through a random-priming amplification procedure (RP-AMP) using primers with a random sequence at the 3' end and a fixed motif at the 5' end. shDNP256 (first strand synthesis): 5'- TAG ATG CTG TTG NNN NNN NNN -3' shT7N9 (second strand synthesis): 5'- ACT ATA GGG AGA NNN NNN NNN -3' 1.5 g of mRNA was reverse-transcribed with Superscript II and the DP256 random primer for 20 minutes at 42 C (10 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 5 U/ l Superscript II). The RNA was degraded with the addition of 20 l volume of 0.5N sodium hydroxide and 0.25 M EDTA for 20 minutes at 65 C. The single stranded cDNA was purified using a commercial kit (Qiagen Qiaquick). The resulting product of single stranded cDNA was placed in its entirety in a second strand reaction. Second strand synthesis reactions utilized shT7N9 random primer and the Klenow fragment of DNA polymerase utilizing standard reaction conditions (37 C for 60 minutes, 0.2 mM DTT, 2.1 mM Tris-HCl pH 7.9, 2.1 mM MgCl2, 10.7 mM NaCl, 1.07 mM dNTPs, 0.1U/ l Klenow), followed by another Qiaquick purification. Multiple polymerase chain reactions were run using 0.15 g of double stranded DNA and standard reaction conditions. Amplification was achieved using 10 cycles of PCR with the DP256 and T7 primers (20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.01 mM dNTPs, 1.5 mM MgCl2, 0.01 U/ l Taq Polymerase) DP256: 5'- GTT CGA GAC CTC TAG ATG CTG TTG -3' T7: 5'- AA TTA ATA CGA CTC ACT ATA GGG AGA -3' followed by Qiaquick purification. Further amplification was achieved using in vitro transcription reactions with X0.5 g dsDNA and T7 RNA Polymerase (7.5 mM DTT, 40 mM Tris-HCl pH 7.5, 14.25 mM MgCl2, 10 mM NaCl, 2 mM Spermidine, 125 U/ml RNAguard, 2.5 mM dNTPs, 15 U/ml IPPase, 25kU/ml T7 Polymerase) for 16 hours at 42 C. The cRNA was purified (RNeasy) and reverse transcribed using Superscript and random 9-mers and amino-allyl dUTP (42 C for 20 minutes, 10 mM DTT, 50mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 0.5 mM aa-dUTP, 5 U/ l Superscript II). The cDNA for each channel was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 1M sodium bicarbonate, pH 9.0, followed by quenching with 4.0 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit and the percentage dye incorporation and total cDNA yield was determined spectrophotometrically. Cy5 and Cy3 labelled cDNAs were combined and added to 2 mls 30% formamide with Herring Sperm DNA for hybridization to the microarray. Further details can be found in Castle et al., Genome Biol 2003;4(10):R66, http://genomebiology.com/2003/4/10/R66 , Roberts CJ et al., Science 2000, 287:873-880, and Schadt, Edwards, et al., unpublished. Normalization of single channel data is described in Ying, et al., "Identification of Chromosomal Regions Containing Transcribed Sequences Using Microarrays and Computational Methods" , 2003 Proceedings of the American Statistical Association, Biopharmaceutical Section [CD-ROM], Alexandria, VA: American Statistical Association, 2003. Briefly, an invariant set of probes was selected based on their intensity ranks. Then, a LOESS regression model was fit for Cy5 vs. Cy3 intensities for all probes in the invariant set. Finally, for all probes on the array the Cy5 intensities were normalized to the corresponding Cy3 intensities using prediction values from the regression model. Note that the normalized intensity values given here were used for all analyses reported in the accompanying paper. The ratio values reported here were not used for the analysis described in the manuscript and are not calculated from the normalized intensity values given below. Keywords: other
2004-03-04 | GSE1097 | GEO
Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.
2022-05-31 | PXD023427 | Pride
Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.
2022-05-31 | PXD023368 | Pride
Project description:S. aureus strain JKD6009 RNase III-HTF and USA300 RNase III-HTF were used in our study to capture the RNA-binding proteome in Staphylococcus aureus. Both strains were inoculated in TSB (Tryptone soya broth, Oxioid CM0129) and overnight cultured in 37°C, 200 rpm shaker. Strain JKD6009 RNase III-HTF was sub-cultured into 190 ml LPM (Coombes et al., 2004) (Low phosphate, low magnesium medium, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 8 µM MgCl2, 1 M KH2PO4, 16 mM Tris-HCl, 0.1% casamino acids, 0.3% Glycerol) and grew from OD600 0.01 to 1. Overnight cultures of USA300 RNase III-HTF was re-inoculated to 65 ml fresh TSB to OD600 0.05 and left to grow to an OD600 of ~3. Cells were then filtered through 0.45 µm filters using a vacuum filtration device (UVO3; (McKellar et al., JoVe 2020; Van Nues et al., Nature Communications 2017)) shifted to same volume of LPM medium for 15 min at 37ºC and UV irradiated in LPM in the Vari-X-linker (λ =254 nm) (https://www.vari-x-link.com; (McKellar et al., 2020; Van Nues et al., 2017)) with an energy dose of 2J/cm2. In total, 6 replicates were collected (three technical and two biological replicates) for each experiment.
2022-05-31 | PXD023408 | Pride
Project description:Parasite lysates were centrifuged (20,000 g, 4°C, 15 min). Beads (blank and with linker attached) were washed 3× with water then twice with lysis buffer. The lysate (~2 mg total protein) was first incubated with 2 mg blank beads for 30 min at 4°C with rotating agitation. The lysate was divided into two then incubated with either 1% DMSO or 100 µM DDD01510706 (competitor) for 30 min at 4°C with agitation. Finally, lysates were incubated with 2 mg of compound-bound beads for 1 h at 4°C with agitation. The beads were then washed 3× with wash buffer (0.8% (w/v) octyl β-D-glucopyranoside, 50 mM Tris pH 8.0, 5 mM EDTA, 1 mg/mL BSA) and 2× Tris-buffered saline (TBS; 50 mM Tris-Cl pH 7.5, 150 mM NaCl). Samples were run 1.5 cm into a Bis-Tris 10% (w/v) acrylamide gel and stained with Coomassie quick reagent for 30 min. The entire gel bands were removed and subjected to in-gel reduction with 10 mM dithiothreitol, alkylation with 50 mM iodoacetamide and digestion with 12.5 μg/mL trypsin (Pierce) for >16 h at 37°C. Recovered tryptic peptides were then vacuum dried prior to analysis
2022-10-15 | PXD033740 | Pride