2.4 RNA Library preparation and Sequencing
Since variation in RNA quality may affect downstream results (Passow et al. 2019), library construction and sequencing were carried out for 81 tissue samples with a RIN value above 8.8 for QuantSeq and a subset of 14 blood samples (for which we also had QuantSeq data) with RIN> 9.4 for NEB (Tables 1 and 2 and data on Dryad). None of these samples showed signs of RNA degradation based on the BioAnalyzer profile(Tables 1 and 2 and data on Dryad ). Whole mRNA libraries (NEB) were made only for 14 selected blood samples with similar RIN and concentration among compared groups (Tables 1 and 2). Library preparation was performed with the NEB Ultra II RNA library prep kit with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). For 3’-end RNA Tag-Seq, library preparation was performed with QuantSeq 3’ mRNA-Seq Library Preparation Kit FWD for Illumina (Lexogen). All procedures were performed according to manufacturer suggested protocols. The quantity and molecular size of the libraries were confirmed by Qubit HS DNA assay (ThermoFisher) and Tapestation 2200 system coupled with High Sensitivity D1000 ScreenTapes (Agilent). Sequencing was performed on Illumina Hiseq X with 150bp pair-end reading for all the QuantSeq samples (Lexogen) and four NEB samples, while the remaining 10 NEB samples were sequenced on a NovaSeq machine (see Results section regarding lack of difference between the NEB samples sequenced with different machines). Raw reads were deposited on NCBI (SRA PRJNA691889, available after manuscript acceptance).