GSM3389561: BAT-Mcu-KO 4C #4; Mus musculus; RNA-Seq
Animals were sacrificed by CO2 asphyxiation followed by cervical dislocation, and tissues were immediately harvested and snap frozen in liquid N2. Frozen BAT samples were homogenized in 1mL Qiazol per 100mg tissue using the Qiagen TissueRuptor II. The homogenate was mixed thoroughly with chloroform (1:5 chloroform:homogenate), incubated for 3 mins at room temperature, and centrifuged at 12,000g for 15 mins at 4C, and 100uL of the resulting aqueous phase was added to 350uL Qiagen buffer RLT plus 250uL of 100% ethanol. The resulting mixture was transferred to a column from the Qiagen RNeasy Mini Kit, and RNA was purified according to the manufacturer's protocol. RNA-sequencing libraries were prepared by the Broad Technology Labs at the Broad Institute based on the Smart-seq2 protocol (S. Picelli, O. R. Faridani, Å. K. Björklund, G. Winberg, S. Sagasser, and R. Sandberg, “Full-length RNA-seq from single cells using Smart-seq2,” Nat. Protoc., vol. 9, no. 1, pp. 171–181, 2014.)