| Metadata |
| datasetIdentifier | PASS01443 |
| datasetType | MSMS |
| submitter | Paolo Soffientini <paolo.soffientini@ifom.eu> |
| submitter_organization | IFOM the FIRC Institute of Molecular Oncology |
| lab_head_full_name | Vincenzo Costanzo |
| lab_head_email | vincenzo.costanzo@ifom.eu |
| lab_head_organization | IFOM the FIRC Institute of Molecular Oncology |
| lab_head_country | Milan, Italy |
| datasetTag | RNA-interactomics |
| datasetTitle | Dux RNA-binding factors |
| publicReleaseDate | 2019-12-31 00:00:00 |
| finalizedDate | 2019-09-10 08:17:52 |
| summary | To identify the potential Dux mRNA-binding factor(s) through which ATR could alter Dux mRNA level, we performed RNA-protein pull-down using a synthetic 3’ untranslated region of Dux mRNA (Dux 3’UTR). After elution, samples were loaded on an SDS-PAGE. The entire gel lane was processed with STAGE-diging protocol. |
| contributors | Paolo Soffientini
Angela Bachi
Sina Atashpaz
Vincenzo Costanzo |
| publication | ATR expands embryonic stem cell fate potential in response to replication stress |
| growth | ESCs were grown in feeder-free culture condition and incubated at 37 °C under 3% O2 tension. ESC medium composed of KnockOut DMEM (ThermoFisher, 10829-018), 15% ESC qualified Fetal Bovine Serum (ThermoFisher, 16141-079), 2 mM L-glutamine, 1/500 home-made leukemia inhibitory factor (LIF), 0.1 mM non-essential amino acids, 0,1 mM 2-mercaptoethanol, 50 units/mL penicillin, 50 mg/mL streptomycin supplemented with the two inhibitors (2i); PD 0325901 (1 µM) and CHIR 99021 (3 µM) was used. MEFs were cultured in high glucose DMEM (Lonza, BE12-614F) supplemented with 10% North American FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 units/mL penicillin, 1 mM Sodium Pyruvate and 50 mg/mL streptomycin (MEF medium). |
| treatment | |
| extraction | |
| separation | Proteins where separated in a 4-20% gradient SDS-PAGE.
Peptide separation was achieved on a linear gradient from 95% Solvent A to 50% Solvent B (80% acetonitrile, 0.1% formic acid) over 20 min and
from 50% to 100% Solvent B in 2 min at a constant flow rate of 0.25 μl/ min on a UHPLC Easy-nLC 1000 (Thermo Scientific), where the LC system was connected to a 25 cm fused-silica emitter of 75 μm inner diameter (New Objective), packed in house with ReproSil-Pur C18-AQ 1.9 μm beads (Maisch) using a high-pressure bomb loader (Proxeon). |
| digestion | Gel pieces were rehydrated with 40 μL of Trypsin(12.5 ng/μL in 100 mM NH4HCO3, after few minutes 60 μL of NH4HCO3 were added and samples were incubated at 37 °C o/n in a commercial tip box filled by water on the bottom to ensure that buffer will not evaporate. |
| acquisition | MS data were acquired using a data-dependent top15 method for HCD fragmentation. Survey full scan MS spectra (300–1750 Th) were acquired in the Orbitrap with 60,000 resolution, AGC target 1e6, IT 120 ms. For HCD spectra the resolution was set to 15,000, AGC target 1e5, IT 120 ms; normalized collision energy 28 and isolation width 3.0 m/z. Two technical replicas of each sample were carried out. |
| informatics | For protein identification the raw data were processed using Proteome Discoverer (version 1.4.0.288, Thermo Fischer Scientific). MS2 spectra were searched with Mascot engine against uniprot_mouse_ database (80894 entries), with the following parameters: enzyme Trypsin, maximum missed cleavage 2, fixed modification carbamidomethylation (C), variable modification oxidation (M) and protein N-terminal acetylation, peptide tolerance 10 ppm, MS/MS tolerance 20 mmu. Peptide Spectral Matches (PSM) were filtered using percolator based on q-values at a 0.01 FDR (high confidence). Proteins were considered identified with 2 unique high confident peptides. Scaffold (version Scaffold_4.3.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at
greater than 95.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. |
| instruments | Q-exactive HF |
| species | mouse |
| massModifications | Static C+57.021464
Variable M+15.9994 |
