Trachipleistophora hominis transcriptome
T. hominis was grown in co-culture with RK-13 cells. Flasks were raised in parallel for 7 days post inoculation, during which time they were trypsinised and split twice to boost levels of infection. Two days after the final trypsinisation the cells were harvested in RNAprotect cell reagent and immediately frozen at -20ºC. Cells suspended in RNAprotect (Qiagen) were thawed and pelleted by centrifugation at 400g. Total RNA was purified from each sample using the standard TRIzol (Invitrogen) extraction protocol, with the addition of a bead beating step (three times for 20 seconds at 5 m/s, with 10 mins incubation on ice between beating). An additional cleanup step using the GeneJet RNA purification kit (Thermo Scientific) was added to remove residual organic solvents from the purified total RNA. The RNA integrity and concentration was assessed using the Agilent RNA 6000 Nano Kit on the Agilent 2100 BioAnalyser. PolyA RNA was isolated from 5µg of purified total RNA. Libraries were prepared using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre Biotechnologies) and sequenced on the Illumina HiSeq 2500 in Rapid-Run mode, producing 100bp single-ended reads, with each library sequenced on two different lanes of the sequencer.