Cells of E. caudatum MZG-1 were collected from a clonal monoculture of E. caudatum that was initially established from a single cell isolated from the rumen of gerenuk [36 (link)]. It was kindly given to us by Dr. Dehority (deceased). This monoculture does not have detectable fungus. Frozen stock cultures of E. caudatum MZG-1 were cryopreserved at − 80 °C and have been used in a number of studies [9 (link), 69 (link), 74 , 75 (link)]. The E. caudatum MZG-1 monoculture was fed a mixed feed containing ground wheat grain, ground alfalfa, and ground grass hays and maintained in SP medium [9 (link)]. The feeding and transfer procedures were conducted under a continuous stream of CO2 to protect the ciliate cells from exposure to oxygen. Total RNA was isolated from an actively growing E. caudatum MZG-1 monoculture after six hours of incubation at 39 °C after transferring to fresh SP medium containing the mixed feed. Total RNA was extracted using the Ribozol RNA extraction reagent (Amresco, Inc., Solon, OH) and then cleaned up using the RNeasy® mini kit according to the manufacturer’s instructions (Qiagen, Inc., Valencia, CA). mRNA was enriched using the Oligo Direct mRNA Mini Kit (Qiagen). One library was constructed for 2 × 100 paired-end sequencing from the mRNA and then sequenced following the manufacturer’s protocol on an Illumina HiSeq 2000 system.
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