title: RNA Sequencing of miR-10b +/+ and miR-10b -/- Mouse Embryonic Stem Cells During Adipocyte Differentiation at Days 0, 12, and 27
design: RNA Sequencing of miR-10b +/+ and miR-10b -/- Mouse Embryonic Stem Cells During Adipocyte Differentiation at Days 0, 12, and 27
library_name: miR-10b-/-.Day27.3_p
library_construction_protocol: 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).