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.