RNA-seq Analysis of Leishmania tarentolae

RNA was isolated using TRIzol (Thermo Fisher Scientific) and resuspended in 10 mM Tris (pH 7), and RNA quality was assessed using the Bioanalyzer 6000 Pico chip (Agilent). mRNA was isolated from 1 μg total RNA using the New England BioLabs (NEB) poly(A) mRNA magnetic isolation module and prepared using the stranded RNA-seq protocol (67 (link)), modified for Leishmania as described previously (68 (link)). Libraries were sequenced on an Illumina HiSeq instrument, obtaining paired-end 150-bp reads. Reads were aligned against our in-house L. tarentolae genome with Bowtie2 (69 (link)) using the “very high sensitivity” parameter or the Geneious assembler (Geneious Prime 11.05) using the “low sensitivity/fastest” option. Differential expression analysis was performed on the Geneious assemblies using the DESeq2 module to compare Tet⁺ and Tet⁻ samples from days 2, 3, 4, 6, and 8 for replicate 1 and from days 2, 3, 4, 5, 7, and 9 for replicate 2. Strand-specific read coverage was calculated directly from BAM files of the Bowtie2 alignments using customized pysam scripts (https://github.com/pysam-developers/pysam).