RNA Sequencing Analysis of miR-10b-5p Knockdown During Brown Adipogenesis
RNA Sequencing Analysis of miR-10b-5p Knockdown During Brown Adipogenesis
Cultured cells were lysed in TRI reagent (Sigma). Immortalised brown and white adipocyte cell lines were generated by culturing the cells from the SVF of iBAT and scWAT of 10-week-old female 129Sv mice with retroviral-mediated expression of temperature sensitive simian virus 40 (SV40) large T-antigen (H-2Kb-tsA58. Cells were cultured in DMEM/F12 medium (Sigma) supplemented with 10% FBS Serum 10500 (Gibco), 1% L-Glutamine and 1% Penicillin-Streptomycin antibiotics (Gibco).  Transfection: Adipocytes were plated in twelve-well plates at densities ranging between 200 and 250 K cells/well in media without antibiotics and transfected with either 50 nM miRNA inhibitors using RNAiMAX (Invitrogen). After 48 hours, adipocyte differentiation was initiated.  Adipocyte differentiation: Plates were transferred to the 37 °C incubator and differentiation was initiated. Induction medium was administered for two days to initiate differentiation. The induction medium consisted of 1 µg/mL insulin, 1 nM 3,3′,5-triiodo-L-thyronine sodium salt (T3), 0.5 mM IBMX, 250 nM dexamethasone, and 125 µM indomethacin. After two days, the induction medium was replaced with maintenance medium, which contained 1 µg/mL insulin and 1 nM T3. Cells were maintained in this medium for the remainder of the experiment, with media changes performed as necessary. RNA was isolated using TRI reagent.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).