RNA isolation and cDNA library preparation

RNA was extracted from frozen tissue using manual grinding and a Qiagen RNeasy micro kit (Hilden, Germany) with the following amendments to protocol: RNA was incubated in nuclease free water for five minutes prior to elution, and this eluate was spun through the same extraction column to maximize RNA yield. RNA concentration was verified using a Nanodrop ND-1000 spectrophotometer (Software version 3.1.2), and quality was confirmed using the Agilent 2100 bioanalyzer (Software version B.02.09.SI720). All cDNA samples were set at the same concentration of the most dilute RNA extraction. Samples were processed using the Illumina TruSeq stranded mRNA LT sample prep kit RS-122-2101 (California, U.S.), and the procedure was followed as described in the low sample protocol. The mRNA from each sample was isolated and purified using AMPure XP magnetic beads (Agencourt; Beverly, Massachusetts) before primary and secondary strand cDNA synthesis. Unique Illumina adapters were ligated, and each sample was PCR amplified before validation. PCR was run for 15 cycles of: 98°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds followed by 5 minutes at 72°C and a final hold at 4°C. Samples were normalized, pooled, and sequenced by the center for applied genetics (TCAG) facilities of the Toronto Sick Kids hospital, Ontario, Canada.