Nanopore Direct RNA-Seq Protocol

The mRNA library preparation followed the SQK-RNA002 kit (Oxford Nanopore)–recommended protocol; the only modification was the input mRNA quantity increased from 500 to 1,000 ng, and all other consumables and parameters were standard. Final yields were evaluated using the Qubit HS dsDNA kit (Thermo Fisher Scientific, Q32851) with minimum RNA preparations reaching at least 200 ng. For all conditions, sequencing was carried out on FLO-MIN106 flow cells using either a MinION MK1C or MinION sequencer. All datasets were subsequently base called (high-accuracy base calling) with a Guppy version higher than 5.0.1 with a Q score cutoff of >7. Long-read alignment was carried out by Minimap2 as previously described^{49 (link)}. Sam files were converted to bam and sorted using Samtools 1.4. Alignments were converted and sorted using Samtools 1.4.1. For the three described samples, Toxoplasma aligned reads range between 600,000 and 800,000. The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935.