mRNA in total RNA was isolated and converted into non-stranded or stranded libraries. Non-stranded libraries were produced manually using reagents provided in the Illumina TruSeq RNA Sample Preparation Kit v2 in accordance with manufacturer’s recommendations. The protocol was modified to produce size-selected libraries by modifying the fragmentation conditions and using a Caliper LabChip XT instrument. Stranded libraries were prepared using a NeoPrep Library Prep System and the reagents provided in the Illumina TrueSeq Stranded mRNA Library Preparation kit. The stranded library prep workflow is similar to the non-stranded workflow, except that it involves additional ribosomal reduction chemistry to maximise the percentage of uniquely mapped reads. Following purification, the RNA was fragmented and synthesised into cDNA using a reverse transcriptase process. The products were then enriched with PCR (maximum 10 cycles) to create the final cDNA library. Enriched libraries were subjected to 75 base paired-end sequencing using Illumina HiSeq 2000 v3 kits following manufacturer’s instructions.
