Whole transcriptome RNA-seq library preparation

Whole mRNA transcriptome library preparation and sequencing was performed using methods as previously described^{35 (link)}. Briefly, RNA sequencing libraries were prepared with TruSeq Stranded mRNA Library Prep Kit (Illumina) using 500 ng – 1 µg RNA. Quality of libraries were validated on an Agilent Bioanalyzer using DNA 1000 reagents and chips (Agilent Genomics, Waldbronn, Germany) to quantify library sizes and confirm the absence of primer dimers. Libraries were quantified using a KAPA Universal Library Quantification kit (Roche Diagnostics Limited) on a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) and library concentrations were adjusted for library size. Pooled libraries of 12–15 pM concentrations were sequenced on a MiSeq System (Illumina) using a MiSeq Reagent Kit v3 (Illumina) with 2 × 75 cycles.