RNA extraction and library preparation

RNA extraction and genomic DNA elimination was carried out using the PAXgene® Blood RNA kit (762164, Qiagen, Feldbachstrasse, Germany) as per the manufacturer’s instructions. Final elution was done into 80 μL RNase-free water. A further RNA precipitation reaction was carried out. Briefly, RNA was resuspended 2.5 × 100% ethanol and 10% sodium acetate and spun at 12,000×g for 30 min at 4 °C. Samples were washed in 75% ethanol. Pellets were air dried and resuspended in 40 μL RNase-free water and total RNA yield was determined by analysis of samples using a TapeStation (Agilent) and Qubit (Thermo Fisher Scientific, Australia). Total RNA was converted to strand-specific Illumina compatible sequencing libraries using the Nugen Universal Plus Total RNA-Seq library kit from Tecan (Mannedorf, Switzerland) as per the manufacturer’s instructions (MO1523 v2) using 12 cycles of PCR amplification for the final libraries. An Anydeplete probe mix targeting both human ribosomal and adult globin transcripts (HBA1, HBA2, HBB, HBD) was used to deplete these transcripts. Sequencing of the library pool (2 × 150 bp paired-end reads) was performed using 2 lanes of an S4 flowcell on an Illumina Novaseq 6000.