RNA Sequencing of miR-10b +/+ and miR-10b -/- Mouse Embryonic Stem Cells During Adipocyte Differentiation at Days 0, 12, and 27
RNA Sequencing of miR-10b +/+ and miR-10b -/- Mouse Embryonic Stem Cells During Adipocyte Differentiation at Days 0, 12, and 27
Cultured cells were lysed in TRI reagent (Sigma). Mouse embryonic stem cells (mESCs) were cultured in Glasgow's MEM (GMEM) (Invitrogen) supplemented with 10% FBS, 2 mM L-Glutamine (Sigma), 0.5% Penicillin-Streptomycin (Gibco), 0.1mM MEM Non-Essential Amino Acids Solution (Gibco), 0.25% Sodium Bicarbonate solution (Gibco, 7.5% stock), 1mM Sodium Pyruvate (Gibco), 0.1mM 2-Mercaptoethanol (Gibco) and 1000units/ml LIF (Merckmillipore) on 0.1% gelatin (Millipore)-coated flasks. mESCs were induced to differentiate into mature adipocytes. Briefly, the cell suspension is prepared at 1 × 10⁵ cells/mL in differentiation medium. 20 μL drops are dispensed onto the inner lid of a 150 mm Petri dish, inverted over the dish, and incubated at 37°C with 5% CO₂ for 48 hours. Embryonic Bodies are collected and plated in a gelatin-precoated 6-well plate. On Day 3, the medium is replaced with 1 μM ATRA and 12.5 μg/mL Ascorbic Acid (AsA), refreshed daily. On Day 7, ATRA is removed, and the medium is replaced with 12.5 μg/mL AsA, 3 nM T3, 0.5 μg/mL insulin, and 0.5 μM rosiglitazone. On Day 12, cells are dissociated with accutase, resuspended in 2 mL differentiation medium, and replated. From Day 15, the medium is changed every 3 days, supplemented with 25 μg/mL AsA, 0.1 μM dexamethasone, 20 μg/mL insulin, 0.5 μM rosiglitazone, 0.06 mM indomethacin, and 0.5 μM IBMX. On Days 21 and 24, the medium is replaced with 25 μg/mL AsA, 20 μg/mL insulin, 0.5 μM rosiglitazone, and 3 nM T3. Adipocyte differentiation is assessed on Day 27 by the presence of lipid droplets. RNA was isolated using TRI reagent.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Library preparation was performed by Novogene (Novogene Co., Ltd). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Library preparation was performed by Novogene (Novogene Co., Ltd).