Illumina NovaSeq 6000 paired end sequencing
RNA sequencing was performed at the National Genomics Infrastructure in Stockholm. A total amount of 500 ng RNA per sample was used as the input material. Libraries were prepared using the Illumina TruSeq Stranded mRNA kit (Illumina Cat. No. 20020595, Illumina, USA) that was adapted for automation on the Aglient NGS Bravo workstation (Agilent Technologies, USA) for a 96-well plate format. Briefly, mRNA was purified from 500 ng of total RNA through selective-binding on poly dT-coated beads, and fragmented using divalent cations under elevated temperature. cDNA was synthesized from the resulting fragments by adding reverse transcriptase SuperScript II Reverse Transcriptase (Cat. No. 1808004, ThermoFisher Scientific). This step was followed by bead clean-up with the AMPure XP solution (ThermoFisher Scientific) to selectively retain fragments of desired lengths. cDNA was then subjected to 3' adenylation, which was followed by adapter ligation to the 3' adenylated end of the fragment. The fragments with ligated adapters were once again cleaned-up on AMPure XP beads to remove non-ligated adapters, followed by PCR to amplify these fragments. The PCR products were purified by binding to AMPure XP beads, washed with 80% ethanol and eluted in EB (Qiagen). The quality of the adapter-ligated libraries was checked on the LabChip GX/HT DNA high sensitivity kit, and concentration by Quant-iT. The libraries with concentrations above 20 nM were normalised and pooled, and the concentration of the final pools were estimated by qPCR.