Qiazol lysis reagent mirneasy micro kit

Total RNA was extracted from sort-purified T cell populations using QIAzol Lysis Reagent/ miRNeasy Micro Kit and from snap-frozen total tissue samples (BAT, scWAT, visWAT), which were previously homogenized using QIAshredder (Qiagen) according to the manufacturer’s instructions. 1 μg total RNA was converted to first strand cDNA using iScript^™ Advanced cDNA Synthesis Kit (Bio-Rad). For cell numbers < 2000 and/or RNA amounts < 200 ng, cDNA synthesis and subsequent amplification was performed using the SMARTer ultra-low input RNA Kit for sequencing – v4 or SMARTer Universal Low Input RNA Kit for Sequencing (Takara Clonetech) according to the manufacturer’s instructions. cDNA was generated in the Thermal Cycler peqStar 2X (Peqlab). Real-time PCR quantification was performed using SsoFast Evagreen Supermix (Bio-Rad) or SYBR® Premix Ex Taq^™ (Takara Clonetech) and gene-specific primer sets on a CFX96 real time system (Bio-Rad). Histone and 18S RNA levels were used for normalization of target gene expression levels. Analysis of candidate genes involved in thermogenesis, browning, lipolysis, glycolysis and inflammation was performed. Primers used for Quantitative Real-Time PCR analyses are listed in Supplemental Table S1.

We extracted total RNA from BMDMs using QIAzol Lysis Reagent (miRNeasy Micro Kit; QIAGEN). The first strand of cDNA was synthesized from 100ng RNA with SuperScript VILO cDNA synthesis kit (Invitrogen), and then RT-qPCR performed on an ABI 7500 Fast real-Time PCR System (Applied Biosystem) using TaqMan universal PCR Master Mix II with UNG. The TaqMan Gene Expression Assay Mix for mouse Cd36 (Mm00432403_m1), Axl (Mm00437221_m1), Tnf (Mm00443258_m1) and Gapdh (Mm99999915_g1) were obtained from Thermo Fisher Scientific. The cycle time values of candidate gene were normalized to endogenous control Gapdh in the same sample. The expression level of mRNA was calculated using delta delta CT (∆∆CT) method as previously described.^{3 (link), 10 (link), 17 (link)}