RNA was prepared after 9 hr of CFB2 RNAi induction in cells expressing VSG2. rRNA was depleted using complementary oligonucleotides and RNaseH as previously described (Minia et al., 2016). It was fragmented, and cDNA libraries were prepared using an Illumina kit before sequencing (MiSeq). Raw reads trimmed then aligned to T. brucei genomes using TrypRNASeq (Leiss et al., 2016) and genomes downloaded from TritrypDB. To assess levels of transcripts from expression sites, reads were aligned to the 2018 version of the Lister427 genome (Müller et al., 2018). Each read was allowed to align once (such that reads for repeated genes are distributed among the copies) and these results are in Supplementary file 3. To obtain more information about annotation of any affected chromosome-internal genes, we repeated the alignment using the well-annotated TREU927 genome (Berriman, 2005; Supplementary file 4). To look for changes in expression, we used DeSeqU1 (Leiss and Clayton, 2016), a user-friendly RStudio DeSeq2 (Love et al., 2014) application. To look for enrichment of particular functional classes, we considered a list of genes in which each individual sequence is present only once (i.e. additional gene copies have been removed) (Siegel et al., 2010). Since, in this list, reads from repeated genes will be under-represented, we multiplied each read count by the gene copy number. Copy numbers were obtained by repeating the alignments, allowing each read to align 20 times.

The distribution of reads across Lister427 chromosomes was visualized using Artemis. For this, we chose induced and uninduced samples with similar total aligned read counts.

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