Comment[ArrayExpressAccession]	E-MTAB-1366
MAGE-TAB Version	1.1
Investigation Title	Differentiation of mesenchymal stem cells into adipocytes.
Comment[Submitted Name]	Differentiation of mesenchymal stem cells into adipocytes.
Experiment Description	Differentiation of mesenchymal stem cells into adipocytes was studied. Control (undifferentiated) stem cells were compared to induced (3 days of induction) cells. In addition, two RNA fractions are studied: RNA associated to polysomes and total RNA.
Experimental Design	development_or_differentiation_design	reference_design	co-expression_design
Comment[AEExperimentType]	RNA-seq of coding RNA
Comment[AEExperimentDisplayName]	RNA-seq of coding RNA of total RNA and polysomal RNA from undifferentiated human mesencyymal stem cells (MSCs) and MSCs induced to differentiate into adipocytes for 72 hours
Experimental Factor Name	cell type
Experimental Factor Type	cell type
Quality Control Type	biological replicate
Public Release Date	2013-07-10
Person Last Name	Spangenberg
Person First Name	Lucia
Person Mid Initials
Person Email	lucia@pasteur.edu.uy
Person Phone	59825220910
Person Address	Mataojo 2020, CP 11400. Montevideo, Uruguay
Person Affiliation	Institut Pasteur de Montevideo
Person Roles	submitter
PubMed ID
Publication Author List	Lucia Spangenberg, Patricia Shigunova,Ana Paula R. Abud, Axel R. CofreM-LM-^A, Marco A. Stimamiglio, Crisciele Kuligovski, Jaiesa Zych, Andressa V. Schittini, Alexandre Dias, Tavares Costa, Carmen K. Rebelatto, Paulo R.S. Brofman, Samuel Goldenberg, Alejandro Correa, Hugo Naya, Bruno Dallagiovanna
Publication Title	Polysome profiling shows extensive posttranscriptional regulation during human adipocyte stem cell differentiation into adipocytes
Publication Status	sumbitted
Protocol Name	P-MTAB-33394	P-MTAB-33395	P-MTAB-33396	P-MTAB-33397	P-MTAB-33398
Protocol Type	growth protocol	nucleic acid extraction protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol
Protocol Description	Stem cells were obtained from adipose tissue from obese human donors ( two males and one female, ages: 41, 52, 23) . All samples were isolated, collected after informed consent had been obtained. hASCs were isolated, cultured and characterized as following. Briefly, 100 ml of adipose tissue was washed with sterile phosphate-buffered saline (PBS) (Gibco Invitrogen). A one-step digestion by 1 mg/ml collagenase type I (Invitrogen) was performed for 30 mins at 37 degrees C during permanent shaking and was followed by filtration through first a 100- and then 40-um mesh filter (BD FALCON, BD Biosciences Discovery Labware, Bedford, MA, USA).	The total and polysomal RNA fractions were extracted by a standard Trizol (Invitrogen) RNA isolation protocol. 	Polysomal fractions were prepared with a modified version of the procedure described by Holetz et al. In brief, hASC cultures at 50 to 60% confluence were treated with 0.1 mg/ml cycloheximide (Sigma-Aldrich) for 10 minutes at 37M-!C. The cells were removed from the culture flasks with a cell scraper and resuspended in 0.1 mg/ml cycloheximide in PBS. The suspension was centrifuged (2000 x g for 5 minutes) and the resulting pellet was washed twice with 0.1 mg/ml cycloheximide in PBS. The cells were lysed by incubation for 10 minutes on ice with polysome buffer (15 mM Tris-HCl pH 7.4, 1% Triton X-100, 15 mM MgCl2, 0.3 M NaCl, 0.1 _g/ml cycloheximide, 1 mg/ml heparin). The cell lysate was centrifuged at 12000 x g for 10 minutes at 4M-!C. The supernatant was carefully isolated, loaded onto 10% to 50% sucrose gradients and centrifuged at 39000 rpm (SW40 rotor, HIMAC CP80WX HITACHI) for 160 minutes at 4M-!C. The sucrose gradient was fractionated with the ISCO gradient fractionation system (ISCO Model 160 gradient former), connected to a UV detector for the monitoring of absorbance at 275 nm, and the polysome profile was recorded.	We subjected total and polysome-associated RNA samples to amplification with the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion), to provide a template for SOLiD libraries. The cDNA libraries were prepared with the SOLiD Whole Transcriptome Analysis Kit and the purified products were evaluated with an Agilent Bioanalyzer (Agilent). Library molecules were subjected to clonal amplification according to the SOLiD Full-Scale Template Bead preparation protocol and sequenced with the SOLiD4 System (Applied Biosystems).	SOLiD Full-Scale Template Bead preparation protocol
Protocol Hardware					AB SOLiD 4 System
Comment[SecondaryAccession]	ERP003461
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/ERR304518-ERR304519,ERR304526,ERR313119-ERR313126
SDRF File	E-MTAB-1366.sdrf.txt
Publication DOI	10.1016/j.scr.2013.06.002