title: GSM4576029: Inguinal adipose tissue, female sucrose (FS8153); Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRI reagent and purified using columns from a PureLink RNA isolation kit. Total RNA samples were sent to Novogene where mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and NEB cDNA libraries were prepared. To construct libraries, first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3'ends of DNA fragments, NEBNext Adaptor with hairpin look structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150-200 bp length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The final library was constructed by PCR amplification and purification of PCR products by AMPure XP beads. Library quality was first determined by Qubit2.0 and the insert size was detected using Agilent 2100. Q-PCR was used to accurately quantify the library effective concentration (>2nM).