8 nt dual indices

RNA was extracted using QIAGEN RNeasy Plus kit (74034). RNA quality was assessed by Agilent RNA ScreenTape on Agilent 2200 Tapestation and quantified by Qubit. The mRNA was enriched by poly-A selection and the second strand synthesis was performed with the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module following manufacturer’s instructions (E6111S). Average final library size was between 380-400 bp. Illumina 8-nt dual-indices were used for multiplexing. Samples were pooled and sequenced on Illumina HiSeq X sequencer as 150 bp paired-end reads with an output of 40 million reads per sample.

Poly(A) + transcripts were isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module kit according to the manufacturer’s protocol (New England BioLabs Inc., Massachusetts, USA). RNA samples were randomly primed (5′ d(N6) 3′ [N = A,C,G,T]) and fragmented. The first strand was synthesized with Protoscript II Reverse Transcription with a modified extension period as to what is stated in the manufacturer’s protocol (30 min at 42 °C). The remaining steps of library generation were carried out according to the manufacturer’s protocol NEBNext^® Ultra^™ II Directional RNA Library Prep Kit for Illumina^® (New England BioLabs Inc., Massachusetts, USA). The quantity and quality of libraries was assessed with Qubit 2.0 (ThermoFisher, Massachusetts, USA) and TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., California, USA), respectively. The final libraries which were roughly 450 bp in size with an insert size of 300 bp, were indexed with Illumina^® 8-nt dual-indices. Libraries were then pooled equimolarly and sequenced on an Illumina^® NovaSeq 6000 S4 (Illumina, California, USA) with a read length configuration of 150 PE for 40 M PE reads per sample (20 M in each direction).