*2.7. RNA Sequencing (RNAseq): Library Preparation and Data Generation*

Six RNAseq libraries were produced according to the manufacturer's protocol (NEB-Next UltraII Directional RNA Library Prep Kit, Illumina, NEB, MA, USA) using 800 ng of total RNA. mRNA pull-up was performed using a Magnetic Isolation Module (NEB, MA, USA). All libraries were mixed in a single tube with equal molarity. The RNAseq data was generated on an Illumina NextSeq500, 75 single-end read, high output mode (Illumina). Quality control was assessed using Fastqc (v0.11.5); reads were trimmed for adapters, low quality 3- , and minimum length of 20 using CUTADAPT (v1.12). STAR aligner (v2.6.0a) was used to align 83 bp single-end reads to an *E. histolytica* reference genome (Entamoeba\_histolytica.JCVI-ESG2-1.0.dna.toplevel.fa) and annotation file (Entamoeba\_histolytica.JCVI-ESG2-1.0.46.gff3), both downloaded from ENSEMBL (strain HM-1:IMSS, imported from the AmoebaDB (https://amoebadb.org/amoeba/app accessed on 28 July 2021)). The number of reads per gene was counted using Htseq-count (v0.9.1) (parameters: -t CDS -i ID -m intersectionnonempty -s reverse).
