qPCR

For human cohort 2 and tissues from mice, reverse transcription, primer design/validation and qPCR were done as follows. RNA was extracted from tissue using RNA STAT60 reagent (Tel-Test, Inc.) or RiboZol reagent (AMRESCO LLC) according to the manufacturer’s instructions. Synthesis of cDNA was done using TaqMan reverse transcription reagents (Thermo Fisher Scientific) using 1 μg of RNA template per reaction, as per manufacturer’s instructions. Transcript expression was then analysed by qPCR in 10 μL duplicate reactions using 1–4 μL of cDNA template. Sybr Green-based qPCR was done using qPCRBIO SyGreen Blue Mix (part number PB20.17-20; PCR Biosystems, UK). Taqman-based qPCR was done using qPCRBIO Probe Blue Mix (part number PB20.27-20, PCR Biosystems, UK). Reactions were loaded into 384-well qPCR plates (part number 72.1985.202; Sarstedt, UK) and run on a Light Cycler 480 (Roche). Transcript expression was calculated based on a cDNA titration loaded on each plate. Expression of each target gene was normalised to expression of 18S rRNA (human gene, RNA18SN5; mouse gene, Rn18s), IPO8 or Ppia, based on consistency of housekeeper expression across all samples. For each transcript, expression is presented relative to the group with the highest expression. Taqman assays (Thermo Fisher) were used to analyse mouse tissues for expression of Ucp1 (cat. no. Mm01244861_m1), Pnpla2 (cat. no. Mm00503040_m1), Lipe (cat. no. Mm00495359_m1) and Dgat2 (cat. no. Mm00499536_m1), and expression of ITGAM (cat. no. Hs00167304_m1) and PTPRC (cat. no. Hs04189704_m1) in SVF and Ads from humans. All other primer sequences are described in Table 2.

Sequences of primers used for qPCR.