title: GSM8680425: Brown adipose tissue, cotadutide, mouse2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated from 30 mg of BAT. Samples were homogenized in 800 ul Trizol, incubated for 5 min and fat layer removed. An additional 200 ul of trizol was added to the homogenate followed by 200 ul 1-bromo-3-chloropropane (BCP). Samples were mixed, incubated at RT for 10 min and centrifuged at 12,000xg for 14 min at 4C. The aqueous phase was transferred to a new tube and 600 ul of 70% ethanol added. Samples were then transferred to an RNeasy Mini spin column, centrifuged at 10,000xg for 15s followed by 700 ul Buffer RW1 and centrifuged. Eighty ul of DNase was added to the column and incubated at RT for 15 min followed by 500ul RPE and centrifuged. RPE wash was performed a second time and the spin column was placed in a new tube. 30 ul of water was applied to the column and centrifuged at 10,000xg for 1 min to elute RNA RNA library preparations and sequencing reactions were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). RNA samples received were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).  RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA).