title: RNA-seq data from a differentiation time course of primary human preadipocytes
design: RNA-seq data from a differentiation time course of primary human preadipocytes
library_name: 0117_28_p
library_construction_protocol: The study was approved by the Ethics Committee from the Capital Region of Denmark (reference H-1-2011-077) and informed consent was obtained from all participants. All experiments were performed in accordance with the relevant guidelines and regulations. This study included a total of five lean controls, five subjects with obesity and T2D according to ICPC-2-DK, and four subjects with obesity and no history of diabetes. All of the individuals included in this study are male. The participants were recruited from Surgical Gastrointestinal Department, Hvidovre Hospital, Denmark. The lean controls were subjects undergoing surgery for laparoscopic inguinal hernia repair. Individuals of both the obese T2D and obese cohorts were subjects to laparoscopic gastric bypass operation. Prior to surgery, all study participants were measured and weighted. Exclusion criteria for all three groups were: alcohol consumption of more than 14 units/week, smoking, daily intake of medicine and presence of chronic/acute diseases. Lean men with diagnosed hypercholesterolemia, hypertension and/or diabetes were excluded. Participants were fasted for at least 12 hrs and blood was drawn before undergoing anesthetics. Blood was analyzed at the Clinical Biochemistry Department, Hvidovre Hospital. Visceral adipose tissue was collected from the omental fat pat with laparoscopic surgery instruments under full narcosis during surgery. The adipose tissue biopsy was immediately rinsed in Phosphate-buffered saline (PBS), minced and digested by collagenase for 2½ hours in 37°C water bath shaking. Digestion was stopped by adding Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% Fetal Bovine Serum (FBS). The suspension was passed through a 200-µm sterile nylon filter (Spectrum Laboratories). The stromal vascular fraction (SVF) from the infranatant and the mature adipocytes from the upper fraction were washed 3 times with DMEM. The SVF was further processed through a 40-µm cell strainer and washed once in DMEM. Cells were plated at 75x106 cells/80 cm2 flask and cultured at 37°C (95% air/5% CO2) in DMEM/F12, 10% (v/v) FBS, 100 U/ml penicillin and 100 mg/ml streptomycin until 3 days prior to induction of differentiation, where FBS was removed from the media. At day 0, cells were differentiated in 5 µg/ml insulin, 10 µg/ml transferrin, 0.2 nM tri-iodothyronine (T3), 1 µM rosiglitazone, 50 µM 3-isobutyl-1-methylxanthine (IBMX) and 1 µM dexamethasone for the first 3 days. Thereafter, IBMX and dexamethasone were removed. Insulin was removed at day 12 and cells were processed at day 15. RNA and DNA were isolated with AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN) according to the manufacturer's protocols.  Quality and yield were assessed by NanoDrop and Qubit dsDNA HS Assay Kit (Life Technologies). For RNA undergoing RNA-seq library preparation, RIN value was determined by Bioanalyzer instrument (Agilent Genomics), using the Agilent RNA 6000 Pico Kit. RNA-sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol (Illumina).. Libraries were sequenced on a NextSeq500 instrument (Illumina) with 38-bp paired end.