title: In-vitro RNAseq of H. defensa AS3-no-APSE rep 3
design: Total RNA was isolated 5 days after inoculating into culture flasks using the mirVana miRNA Isolation Kit (Ambion, Thermo Fisher Scientific) followed by DNase treatment using the TURBO DNA-free Kit (Ambion, Thermo Fisher Scientific) and ethanol precipitation in the presence of glycogen.  A total of two (A2C) or three (AS3, ZA17) separate cultures per strain were extracted generating 2 or 3 independent biological replicates.  We sequentially depleted rRNA using the Ribo-Zero (Bacteria) Magnetic Kit (Epicentre, Illumina) according to manufacturerÕs protocol followed by Ribo-Zero-Treated RNA purification using the Illumina modified RNeasy MinElut Cleanup Kit (Qiagen).  RNA templates were quality checked by the Georgia Genomics Facility using an Agilent 2100 Bioanalyzer (Agilent Technologies), Fragment Analyzer (Advenced Analytical).  Standed, paired-end sequencing libraries (75 bp) were generated using the Kapa stranded RNA-seq Library Preparation Kit (Kapa Biosystems) and Illumina sequenced on a NextSeq (150 Cycles) Mid Output Flow Cell