title: GSM8371761: BKO2; Mus musculus; RNA-Seq
library_construction_protocol: For isolation of total RNA from mouse tissue, the entire adipose tissue was homogenized in 1 mL Trizol (Life Technologies) and ceramic beads (Mobio, Dianova GmbH) with a FastPrep homogenizer (MP Biomedicals) at 5500 rpm for 2.~ 20 sec. After 5 min incubation at RT, 200 μL chloroform were added and mixed for 15 sec, before centrifugation at 12000 g for 15 min at 4 °C was performed. The aqueous upper phase containing the RNA was collected and precipitated with 500 μL isopropanol (10 min incubation at RT, following centrifugation at 12000 g for 10 min, 4°C). RNA pellet was washed with 75% ethanol (5 min centrifugation at 7500 g, 4 °C) and briefly dried. Finally, the RNA pellet was resuspended in 50 μL ultra-pure H2O and incubated at 60°C for 10 min. The RNA concentration and purity was determined with a NanoDrop ND-1000 UV-Vis spectrophotometer (Peqlab). Before cDNA synthesis, 10 μg RNA was digested with DNase I (New England Biolabs). The digestion mix (10 μg RNA, diluted 10X digestion buffer and 1 μL DNase I in a total volume of 20 μL) was incubated 10 min at 37°C. Afterwards, heat inactivation of DNase I was performed at 75°C for 10 min. RNA concentration was redetermined and the samples were stored at −80°C. Illumina TruSeq Stranded mRNA