Nanopore library followed the SQK-RNA002 kit (Oxford Nanopore) recommended protocol, the only modification was the input mRNA quantity increased from 500 to 1000 ng, all other consumables and parameters were standard. Final yields were evaluated using the Qubit HS dsDNA kit (Thermofisher Q32851) with minimum RNA preps reaching at least 150 ng. For all conditions, sequencing was performed on FLO-MIN106 flow cells either using a MinION MK1C or MinION sequencer running Minknow v20.06.5 and guppy v4.09. Basecalling was performed during the run using the fast-basecalling algorithm with a Q score cutoff >7. Long read alignment (to ME49-Toxodb-13 and TAIR10 reference fasta files) was performed using Minimap2 (ver 2.1) with the following parameters: ‘-ax splice -k14 -uf -G 5000 t 10 --secondary=no –sam-hit-only’ for Toxoplasma and ‘-ax splice -k14 -uf -G 20000 t 10 --secondary=no –sam-hit-only’ for Arabidopsis. Aligned reads were converted to bam, sorted, and indexed using Samtools. For T. gondii datasets, most sequencing runs were stopped after having generated between 400 k and 500 k of aligned reads to keep a standard of comparison (T. gondii reads varying between 30% and 70% of total mRNA depending on the preparation).

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