GSM9886073: inguinal white adipose tissue, Long-term lean control, biological replicate 3, mouse SMA_3758; Mus musculus; RNA-Seq
Unless otherwise stated, all steps were performed on ice. Tissues were homogenized at room temperature in 500 µL TRI Reagent® (Sigma-Aldrich, T9424) and six 1.4 mm ceramic beads (OMNI International, 19-645) per 80 mg tissue using the mouse liver program on a FastPrep-24TM 5G (MP Biomedicals). Homogenates were centrifuged at 10,000 x g for 10 min at 4 °C, and the infranatant was transferred to a new DNA LoBind tube (Eppendorf), avoiding the lipid layer and debris pellet. 0.2 volumes of chloroform (Acros Organics, 383770025) were added, samples were mixed by inversion, incubated at room temperature for 5 min, and centrifuged at 10,000 x g for 10 min at 4 °C. The aqueous phase was transferred to a new DNA LoBind tube. 0.6 volumes of isopropanol (VWR Chemicals, 20880.320) were added, samples were mized by inversion, incubated at -20 °C for 1 hr, and centrifuged at 12,000 x g for 15 min at 4 °C. The supernatant was removed, and the pellet was washed with 1mL 70% ethanol (VWR Chemicals, 20824.365) prepared in DEPC-treated water (Sigma-Aldrich, D5758), followed by centrifugation at 12,000 x g for 10 min at 4 °C. Illumina libraries were prepared using poly(A) enrichment according to the manufacturer's instructions and sequenced with paired-end reads on a NovaSeq 6000 (Illumina).