Illumina compatible libraries were prepared from RNA using QuantSeq 3′ mRNA-seq Library Prep Kit FWD for Illumina (Lexogen GmbH, Wien, Austria) according to the manufacturer’s instructions. In brief, library generation was initiated by oligo-dT priming and first-strand synthesis. After RNA removal, libraries were subjected to random-primed second-strand synthesis. Illumina specific linker sequences are added by the primer, and the resulting double-stranded cDNA purified with magnetic beads. An additional 12 cycles of PCR amplification were carried out in order to introduce barcodes and to generate sufficient amounts of DNA required for cluster generation. After final purification, libraries were measured on TapeStation and Qubit (ThermoFisher, Waltham, MA) to determine quantity and size. The resulting libraries were on average 400-bp size with an average insert size of 270 bp. The method does not require prior poly(A) enrichment or ribosomal RNA depletion. ERCC (External RNA Controls Consortium) RNA Spike-In Mix (Cat# 4456740 Thermo Fisher Scientific, Waltham, MA) was added to the RNA before library preparation to allow inter-sample normalization and control for variabilities.
