GSM8658381: iWATirHFD_3; Mus musculus; RNA-Seq
Total RNA was isolated from inguinal fat or epididymal fat tissues using TRIzol reagent (Invitrogen, 15596018) and RNeasy Mini purification kit (Qiagen, 74104) according the manufacture's protocol. Tissues were homogenized in TRIzol reagent using a bead homogenizer for 20 min at max speed (Qiagen, TissueLyser II). DNA was digested on column using RNase-Free DNase Set (Qiagen, 79254). For adipose tissue RNAseq, RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, and then the second strand cDNA synthesis was either the same way as the first strand (for non strand specific library) or was synthesized using dUTP, instead of dTTP to generate the directional library (for strand specific library). The library was ready after end repair, A-tailing adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. After library quality control, different libraries were pooled based on the effective concentration and targeted data amount, then subjected to Illumina sequencing