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 only for 81 tissue samples with a RIN value above 8.8 for 3’ Tag-Seq and for 14 samples with RIN > 9.4 for whole mRNA-Seq (Supporting Information Table S1). None of these samples showed signs of RNA degradation based on the BioAnalyzer profile. Liver samples were excluded from the subsequent processing as most of the samples had a low RIN and we therefore did not have more than 3 samples per group per comparison (Supporting Information Table S1). Whole mRNA libraries (NEB) were made only for selected blood samples with similar RIN and concentration among compared groups (see below and Supporting Information Table S1 for additional information). 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) (see Ma et al., 2019 for further details on differences between these two RNA-Seq methods). 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 3’ Tag-Seq 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 will be deposited to NCBI after manuscript acceptance.