RNA-Seq of Amoebophilus asiaticus
Ribosomal RNA was removed with the RiboZero Magnetic kit as recommended by the manufacturer and in addition RNA from symbiont-free Acanthamoeba was treated with the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific). Quality control of purified RNA was performed using the Experion Automated Electrophoresis Station (Bio-Rad).  RNA fragmentation was performed at 70C for 5 min (RNA fragmentation reagents, Thermo Fisher Scientific). The NEBNext Ultra Directional RNA Library Prep Kit (Illumina) and NEBNext Multiplex Oligos (New England Biolabs) were used for first-strand cDNA synthesis for a strand-specific cDNA library preparation. Libraries were sequenced on an Illumina HiSeq2000 machine with each library on an individual lane.