Illumina HiSeq 2500 sequencing; RNA-seq of total RNA from human adipose tissue stem cells induced to adipogenesis and osteogenesis for 24 hours
RNA-seq of total RNA from human adipose tissue stem cells induced to adipogenesis and osteogenesis for 24 hours
All samples were isolated, collected after informed consent had been obtained, in accordance with guidelines for research involving human subjects, and with the approval of the Ethics Committee of Fundação Oswaldo Cruz, Brazil (approval number 1.308.195). hASCs were isolated of 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 min at 37 °C during permanent shaking and was followed by filtration through first a 100- and then 40-μm mesh filter (BD FALCON, BD Biosciences Discovery Labware, Bedford, MA, USA). The cell suspension was centrifuged at 800 g for 10 min, and contaminating erythrocytes were removed by erythrocyte lysis buffer, pH 7.3. The cells were washed and then cultivated at a density of 1 × 105 cells/cm2 in T75 culture flasks in DMEM-F12 (Gibco Invitrogen) supplemented with 10% FCS, penicillin (100 units/ml), and streptomycin (100 μg/ml). The medium was changed 2 days after the initial plating. The culture medium was then replaced twice each week. ASCs were subcultured after the cultures had reached 80% to 90% confluence; cells were detached by treatment with 0.25% trypsin/EDTA (Invitrogen) and were replated as passage-1 cells (the process was then continued). The characterization of the cells has been done following the minimal criteria for defining multipotent mesenchymal stromal cells as determined by the International Society for Cellular Therapy (Dominici et al., 2006). All tests were performed with cell cultures at passages 3 to 5. Polysomal, monosome and free fractions from adipose tissue stem cells cultures treated with differentiation medium for 24 hours were prepared according to (Spangenberg et al., 2013). In brief, cells were treated with 0.1 mg/mL cycloheximide (Sigma-Aldrich) for 10 min at 37oC, removed from the culture flasks with a cell scraper, and resuspended in 0.1 mg/mL cycloheximide in PBS. The suspension was centrifuged (2,000 g for 5 min), and the resulting pellet was washed twice with 0.1 mg/mL cycloheximide in PBS. The cells werelysed by incubation for 10 min on ice with polysome buffer (15mM Tris-HCl pH 7.4, 1% Triton X-100, 15mM MgCl2, 0.3M NaCl, 0.1 mg/mL cycloheximide), and the cell lysate was centrifuged at 12,000 g for 10 min at 4oC. The supernatant was carefully isolated, loaded onto 10% to 50% sucrose gradients, and centrifuged at 39,000 rpm (HIMAC CP80WX HITACHI) for 160 min at 4oC. The sucrose gradient was fractionated with the ISCO gradient fractionation system (ISCO Model 160 gradient former), connected to a UV detector to monitor the absorbance at 275 nm, and the polysome profile was recorded. The Total, free, monosome and polysomal RNA fractions were extracted by DirectZOL (Zymo Research).  For adipogenic differentiation, hASCs were treated for 24 hours with hMSC Adipogenic Differentiation Bullet Kit (Lonza - PT-3004) and hMSC Osteogenic Bullet Kit (Lonza - PT-3002) , in accordance with the manufacturer's instructions. The RNA from total, free, monosome and polysome fractions was extracted using DirectZol kit (Zymo). Illumina® TruSeq® Stranded Total RNA Sample Preparation kits
FIOCRUZ - PARANA - Instituto Carlos Chagas
E-MTAB-6298:TL10_ADI_Total_s