title: GSM8213523: iWAT, TAN1_12PM; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using the Trizol Reagent (Invitrogen). The aqueous phase containing the RNA was loaded onto a gDNA Eliminator Spin Column (Qiagen, USA) for elimination of genomic DNA, and RNA was isolated using the RNeasy Plus kit (Qiagen) according to manufacturer's instructions. Total RNA (500 to 1000 ug) was reverse transcribed into cDNA using the M-MLV reverse transcriptase (Invitrogen), 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, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.