title: RNA-seq of visceral adipose tissue in obesity patients(sRNA)
design: Briefly, 1.5 g RNA samples were used as input material for rRNA removal using Ribo-Zero rRNA Removal Kits (Epicentre, Madison, WI, USA). Sequencing libraries were generated with Next Ultra Directional RNA Library Prep kits  (Illumina, New England Biolabs, USA) following the manufacturers instructions. First-strand cDNA was synthesized using random hexamer primer and reverse transcriptase. After adenylation of the 3 ends of DNA fragments, Nebnext hairpin loop structure adapters were ligated to prepare for hybridization. The library fragments were purified with AMPure XP Beads (Beckman Coulter, Beverly, MA, USA) to select insert fragments of 150200 bp in length. The cDNA was treated with 3 L uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII (USER) enzyme (New England Biolabs, USA) at 37C for 15 min before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index(X) Primer. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform at Biomarker Technologies (Beijing, China), and paired-end reads were generated.