Whole Transcriptome
Samples are run on the Agilent Tapestation 4200 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. One microgram of total DNAse treated RNA is then prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina.
Total RNA is depleted of its rRNA 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 AmpureXP beads, then validated again on the Agilent Tapestation 4200. Before being normalized and pooled, samples are quantified by Qubit then run on the Illumina NextSeq 2000.