Samples are run on the Agilent 2100 Bioanalyzer to determine level of degradation thus ensuring only high quality RNA is used (RIN Score 8 or higher). The Qubit fluorometer is used to determine the concentration prior to staring library prep. 4 µg of total DNAse treated RNA are then prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. Poly-A RNA is purified and fragmented before strand specific cDNA synthesis. cDNA are then a-tailed and indexed adapters are ligated. After adapter ligation, samples are PCR amplified and purified with Ampure XP beads, then validated again on the Agilent 2100 Bioanalyzer. Samples are quantified by Qubit before being normalized and pooled, then run on the Illumina HiSeq 2500 using SBS v3 reagents.