RNA-Seq of human floated adipocytes from subcutaneous adipose tissue: Sample RNA-Seq_Hs_SAT_IR_211-1
Whole tissue subcutaneous adipose specimens were freshly collected from the operating room. Skin was removed, and adipose tissue was cut into 1- to 2-inch pieces and rinsed thoroughly with 37C PBS to remove blood. Cleaned adipose tissue pieces were quickly minced in an electric grinder with 3/16-inch hole plate, and 400 ml of sample was placed in a 2-l wide-mouthed Erlenmeyer culture flask with 100 ml of freshly prepared blendzyme (Roche Liberase TM, research grade, cat. no. 05401127001, in PBS, at a ratio of 6.25 mg per 50 ml) and shaken in a 37C shaking incubator at 120 r.p.m. for 15–20 min to digest until the sample appeared uniform. Digestion was stopped with 100 ml of freshly made KRB (5.5 mM glucose, 137 mM NaCl, 15 mM HEPES, 5 mM KCl, 1.25 mM CaCl2, 0.44 mM KH2PO4, 0.34 mM Na2HPO4 and 0.8 mM MgSO4), supplemented with 2% BSA. Digested tissue was filtered through a 300 μM sieve and washed with KRB/albumin and flow through until only connective tissue remained. Samples were centrifuged at 233g for 5 min at room temperature, clear lipid was later removed, and floated adipocyte supernatant was collected, divided into aliquots and flash-frozen in liquid nitrogen. Total RNA from ~400 μl of thawed floated adipocytes was isolated in TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For RNA-seq library construction, mRNA was purified from 100 ng of total RNA by using a Ribo-Zero rRNA removal kit (Epicentre) to deplete ribosomal RNA and convert into double-stranded complementary DNA by using an NEBNext mRNA Second Strand Synthesis Module (E6111L). cDNA was subsequently tagmented and amplified for 12 cycles by using a Nextera XT DNA Library Preparation Kit (Illumina FC-131). Sequencing libraries were analyzed with Qubit and Agilent Bioanalyzer, pooled at a final loading concentration of 1.8 pM and sequenced on a NextSeq 500.