Illumina NovaSeq 6000 paired end sequencing; Bulk RNAseq of Mus musculus Protein Kinase D1 (Prkd1) knock-out adipose tissue in response to thermoneutral and cold exposure stress
The interscapular brown adipose tissue (iBAT) was harvested from mice following euthanasia using carbon dioxide at 5 liters per minute for 3 minutes. Prkd1 WT or brown adipose tissue KO (background: C/57Bl6) mice were maintained on chow diet and acclimated at thermoneutrality for 2 days (30 degrees Celsius) followed by either 8 hours or 4 days at 6 degrees Celsius in the Powers temperature regulated cabinet. iBAT was harvested and placed in 350 uL Trizol solution. The sample was prepared according to the Trizol User Reagent Guide through step 8. Following Trizol extraction, samples were placed on Qiagen RNA extraction columns according to the manufacturer's protocol until purified RNA was obtained. RNA was amplified using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara), and cDNA libraries were constructed using the Low Input Library Prep Kit (Takara).