Table 3.

Short RNA samples used in this work

Sample	Total number of read pairs	Merged and trimmed	Mapped to nuclear genome (%)	Mapped to maxicircle (%)	Mapped to minicircles (%)
BSF+PCF, replicate 1	148 935 513	143 870 651	117 351 028 (81.6)	341 908 (0.24)	3 476 778 (2.42)
BSF, replicate 2	79 132 567	52 260 258	41 163 148 (78.8)	122 246 (0.26)	3 008 890 (5.76)
PCF, replicate 2	85 827 714	40 535 598	31 920 884 (78.7)	164 732 (0.33)	490 364 (1.21)

Notes: PCF cells had been obtained by differentiation of an aliquot of the same T. brucei EATRO 1125 BSF culture that had been used for preparing RNA for that life cycle stage. Total RNA was isolated from crude mitochondrial fractions, short RNAs (<200 nt) were enriched using the PureLink miRNA isolation kit (ThermoFisher) and barcoded libraries prepared using the Illumina Small RNA kit. Although a single read was expected to cover an entire gRNA sequence, paired 125-bp reads were generated as they offered the opportunity to produce merged reads of improved accuracy. Inadvertently, BSF and PCF libraries for technical replicate 1 had been prepared using adapters with the same barcode and sequenced in the same lane and could therefore not be distinguished (both replicates used RNA isolated from the same crude mitochondrial fraction, but size selection, library preparation and sequencing were carried out separately). Reads for contaminating nuclear RNA were removed by mapping with relaxed stringency to the T. brucei TREU927 reference genome (34), version 5.2 (downloaded from www.TriTrypDB.org). Remaining reads were then mapped with high stringency to a T. brucei EATRO 1125 maxicircle sequence (assembled as part of this project) and to the 391 assembled minicircles. See Materials and Methods for more technical details.