RNAseq of mus musculus ob/ob liver tissue
Total RNA samples treated with RNase-free DNase I (Qiagen, Valencia, CA) were quantified and analyzed using Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham MA) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using Illumina TruSeq RNA Library Prep Kit v2 according to the manufacturers instructions (Illumina, San Diego, CA). Briefly, poly(A)-containing mRNA in 150 ng total RNA was converted into a double-stranded cDNA library which was, in turn, ligated to TruSeq paired-end adaptors containing unique index barcode sequences. The ligated products were purified and enriched with 15 cycles of Polymerase Chain Reaction (PCR). The size distribution of the RNA-seq libraries was analyzed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and the library concentrations were determined using Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA). Sequencing libraries were normalized and pooled to 4 nM in 10 mM Tris-HCl, pH 8.0 and subjected to denaturation in 0.1 N NaOH for 5 min prior to the final dilution to 1.8 pM. Denatured and diluted libraries were loaded onto NextSeq 500/550 v2 flow cells and paired-end sequencing (2 x 75 cycles) was performed on NextSeq 500 sequencing instruments (Illumina, San Diego, CA).