title: GSM8511666: iWAT, HFD, male, WT, rep4; Mus musculus; RNA-Seq
library_name: GSM8511666
library_construction_protocol: At the end of the feeding period, mice were food deprived for 4 hours and anesthetized with ketamine/xylazine/acepromazine (80:100:150 mg/kg, intraperitoneally, respectively), and tissues collected. Mice were euthanized under anesthesia. Inguinal white and interscapular brown adipose tissue (iWAT and iBAT, respectively) depots and the liver from mice fasted for 4h were treated with RNAlaterTM stabilization solution (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA) upon excision before snap freezing in liquid nitrogen (N2) and storage at -80oC. Total mRNA was extracted from iWAT and iBAT depots and the liver using the PureLinkTM RNA Mini Kit (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA). All tissue samples were first homogenized using TRIzolTM reagent (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA) for total RNA isolation. RNA abundance and quality were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Total cellular mRNA extracts derived from the iWAT of WT and LFABP-/- mice were submitted for RNA-Sequencing (RNA-Seq) analysis to Rutgers Genomics Center (Rutgers University, Newark, NJ). RNA quality was first checked for integrity using an Agilent 2200 TapeStation (Agilent Technologies); samples with RNA integrity number (RIN) >7.0 were used for subsequent processing. Total RNA was subjected to two rounds of poly(A) selection using oligo-d(T)25 magnetic beads (New England Biolabs). Illumina compatible RNA-seq library was prepared using a NEB next ultra RNA-seq library preparation kit. The cDNA libraries were purified using AmpureXP beads and quantified on an Agilent TapeStation and Qubit 4 Fluorometer (Thermo Fisher Scientific). An equimolar amount of barcoded libraries was pooled and sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA) using the 1X100 cycles configuration.