GSM2776547: BAT rep1; Mus musculus; OTHER
Small pieces (50–100 mg) of frozen brown, inguinal white and epididymal white adipose tissues were Dounce homogenized in 4 mL homogenization buffer. After centrifugation at 13,000 rpm for 10 min, the lipid layer was removed and the supernatant was collected and incubated with anti-GFP antibody (5 mg/mL; Abcam, ab290) for 1 hr at 4 ˚C. Protein G dynabeads were washed twice in low-salt wash buffer, added to the homogenates with antibody, and subsequently incubated for 30 min. Dynabeads with immunoprecipitates were washed three times in high-salt wash buffer. Following the last wash, RLT buffer with b-mercaptoethanol was added to dynabeads, and RNA was extracted using a QIAGEN Micro RNeasy kit per the manufacturer’s instructions. Extracted RNA (100 ng) was processed for rRNA removal using the Epicenter rRNA depletion kit per the manufacturer’s instructions. rRNA-depleted RNA was subsequently used to generate single-end sequencing libraries using the Illumina RNA TruSeq Library Kit per the manufacturer’s instructions.