Relative Quantification of Gene Expression

Cell Total RNA isolation kit (FOERGENE, Chengdu, China) was used to extract total RNA, and then reverse-transcribed into cDNA by Reverse transcription kit (ABclonal, Wuhan, China). Genious 2x SYBR Green Fast qPCR mix (ABclonal, Wuhan, China) was used to perform qPCR and detected by Bio-Rad CFX96 qPCR instrument (Bio-Rad, Hercules, CA, USA). In a previous study, we found that PFDN5 and TBP genes were the most stable reference genes for BAT to WAT transformation through RNA-seq [35 (link)]. Thus, TBP and PFDN5 genes were applied as reference genes. Primers used in this study were shown in Table S1. The relative expression levels of the target genes were normalized relative to the expression of PFDN5 and TBP using the 2^−ΔΔCT method. Six biological replicates and three technical replicates in a biological sample were used for qPCR.