S5
Total RNA was assessed for purity and size integrity using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, USA). Purification of messenger RNA (mRNA) was performed using oligo-dT beads provided in the NEBNExt Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA). Complementary DNA (cDNA) libraries for Illumina sequencing were constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) and NEBNext Mulitplex Oligos for Illumina using the manufacturer-specified protocol. Briefly, the mRNA was chemically fragmented and primed with random oligos for first strand cDNA synthesis. Second strand cDNA synthesis was then carried out with dUTPs to preserve strand orientation information. The double-stranded cDNA was then purified, end repaired and a-tailed for adaptor ligation. Following ligation, the samples were selected for a final library size (adapters included) of 400-550 bp using sequential AMPure XP bead isolation (Beckman Coulter, USA). Library enrichment was performed and specific indexes for each sample were added during the protocol-specified PCR amplification. The amplified library fragments were purified and checked for quality and final concentration using an Agilent 2200 Tapestation (D1000 chip, Agilent Technologies, USA) combined with a Qubit fluorometer (ThermoFisher, USA). The final quantified libraries were pooled in equimolar amounts for clustering and sequencing on an Illumina NovaSeq 6000 DNA sequencer, utilizing a 50 bp paired-end cycle sequencing kit on an S2 flowcell (Illumina, USA). The software package Real Time Analysis (RTA), was used to generate raw base call files (bcl), which were then de-multiplexed by sample into fastq files using bcl2fastq Conversion Software v2.20 (Illumina, USA).