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.