RNA-Seq of mus musculus treated with fenofibrate: adult retina
Whole retina was isolated from eight 6-month-old C57BL/6J mice receiving a fenofibrate-supplemented diet for 3 months, and eight sibling controls (with four male and four females per group). RNA was isolated using Trizol, RNA concentration was determined by Qubit, and RNA quality was assessed with an Agilent 2100 Bioanalyzer Total RNA nano chip (Agilent Technologies Inc., Santa Clara, CA, USA). Library preparation was performed with 1 ug of total RNA. Ribosomal RNA was removed by a hybridization method using the Ribo-ZERO kit (Illumina, San Diego, CA, USA). Depletion and mRNA yield were confirmed with an Agilent 2100 Bioanalyzer as above. Messenger RNA was then fragmented in buffer containing 40 mM Tris Acetate pH 8.2, 100 mM Potassium Acetate and 30 mM Magnesium Acetate and heating to 94 C for 150 s. Messenger RNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (per manufacturers instructions, Life Technologies, Carlsbad, CA, USA) and random hexamers. A second strand reaction was performed to yield ds-cDNA. Complimentary DNA was blunt ended, had an A base added to the 3 ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 1215 cycles using primers incorporating unique index tags. Libraries were quantified using Qubit assays and quality assessed on an Agilent TapeStation system (Agilent Technologies, Santa Clara, CA, USA). An equimolar pool was made of all libraries with unique indices. Fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases (Illumina, San Diego, CA, USA).