Dnase 1 treatment

The extraction was performed as previously described [8 (link)]. Briefly, cell lysis and RNA isolation was done with the TRIzol Plus RNA purification kit (AMbion). 5 mL TRIzol reagent was added to the frozen pellet followed by 1 mL chloroform after cell lysis. After shacking and centrifugation, the upper phase was mixed with an equal volume of 70% ethanol before transfer to PureLink RNA Spin Cartridges furnished with the kit. Kit instructions allowed to obtain purified total RNA which was further processed by cleanup with the RNeasy mini kit (Qiagen). DNA was eliminated with DNAse I treatment (AM1906, Invitrogen). 20 μL RNA were treated with 2 μL DNAse in 50 μL nuclease free water for 30 min at 37 °C before addition of another 1.5 μL DNAse and 30 min extra incubation. Treatment was done twice and was followed by RNA cleanup with the RNeasy kit. A full quality assessment of the RNA, quantification using Nanodrop, PCR to check for residual DNA and migration on 1% agarose gel were done before depletion of ribosomal RNA with MicrobExpress kit (ThermoFischer). Depleted RNA samples were quantified and stored at − 80 °C before being sent for analysis. DNA libraries from RNA samples and Hi-seq single read sequencing were performed by the Imagif platform (I2BC, Orsay, France).

Total mRNA was extracted with Trizol (Invitrogen, USA) and purified by RNeasy Lipid Tissue Mini Kit (Qiagen, USA), followed by DNase I treatment (Invitrogen, USA). Total mRNA was reverse-transcribed using the Iscript cDNA kit (Bio-Rad, USA). Real-time PCR was performed using a StepOne Plus real time PCR system (Applied Biosystems, USA) and the QuantiTect primers (Qiagen, USA) QT00291151, QT00149240, QT00258692, QT00253967, QT01195901, QT00156303, QT01044953, QT01044946, QT00097300, QT00249452, QT00249732, QT00296044 and QT01062656 against mouse stearoyl-CoA desaturase (Scd1), fatty acid synthase (Fasn), adrenoceptor beta 1 (Adrb1), adrenoceptor beta 2 (Adrb2) and adrenoceptor beta 3 (Adrb3), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), glucose transporter 1 (Slc2a1) and 4 (Slc2a4), uncoupling protein 1 (Ucp1), type 2 iodothyronine deiodinase (Dio2), G-protein coupled receptor 3 (Gpr3), G-protein coupled receptor 6 (Gpr6) and G-protein coupled receptor 12 (Gpr12), respectively. Normalization was performed with the QuantiTect primer assay against mouse Rpl19 (QT01779218) housekeeping gene.

Cells were lysed using the TRIzol reagent (Invitrogen). Brains were removed from euthanized mice and frozen in liquid nitrogen. RNA was extracted using the TRIzol reagent (Invitrogen) according to manufacturer. After DNaseI treatment (Invitrogen) RNA from cells and brains was reverse transcribed into cDNA with oligo d(T) primer and OMNISCRIPT RT KIT (Qiagen). QPCRs were performed as described (Piersanti et al., 2015 (link)) using the following primers:

The TRIzol reagent (Invitrogen) was used to extract total RNA. Potential genomic DNA contamination was eliminate by DNase I treatment (Invitrogen). The synthesis of cDNA was conducted using the PrimeScript reverse transcriptase reagent kit (TaKaRa). Quantitative RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) on the Applied Biosystems 7500 (Life Technologies). The Rpl7 gene served as a reference gene for normalization. Primer sequences used in this study were listed in Supplementary Table S1.

RNA was isolated from snap-frozen lumbar DRG using the Trizol method (Invitrogen) and purified using DNAseI treatment (Invitrogen) as per manufacturer’s instructions. For cDNA synthesis, 1 µg of total RNA was diluted in cDNA Master Mix using reagents from cDNA synthesis kit (Applied Biosystems). Relative expression of target genes was measured by qRT-PCR and normalized with the expression of 18srRNA, using the Lightcycler 480 real-time PCR system (Roche). Primer sequences are given in Supplementary Table T1.