RNA extraction
Sample tissues were mechanically disrupted prior to RNA isolation. Approximately 30 acid washed glass beads (Sigma, 710-1180 µm diameter) and 600 µL RLT buffer (Qiagen) were added to each sample. The samples were then subjected to 2 x 40 s cycles of bead beating at 4/s in a fast Prep-245G (MP Biomedicals). Total RNA was isolated from the sample homogenate using Qiagen RNeasy mini kit (including the optional DNase treatment). Total RNA was eluted in 40 µL of RNase free water and 3 µL were visualized on a 1% agarose, 0.5 x TBE gel for quality check. RNA concentration was measured using the Qubit RNA HS Assay (Thermo Fisher Scientific/Invitrogen), with fluorescence analysis on a NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scientific). Between 500 pg and 100 ng total RNA underwent reverse transcription and cDNA was amplified using NuGen’s Ovation V2.0 kit (with one cycle amplification). The amplified cDNA was then purified using magnetic beads (Beckman Coulter Agencourt kit) and 1 µL was visualized on a 1% agarose, 0.5 x TBE gel. Purity of sample cDNA was determined by A260/A280 ratios measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). cDNA concentration was measured using the Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific/Invitrogen). Sample cDNA concentrations were normalized and 25 µL of 20 ng/µL cDNA were sent to Ramaciotti Centre for Genomics (UNSW, Sydney) for Nextera XT Library Preparation and paired-end sequencing on the Illumina NextSeq platform.