GSM9194480: Eimeria acervulina Sporulated 4 months replicate 3; Eimeria acervulina; RNA-Seq
Total RNA was extracted from frozen oocyst Trizol pellets (unsporulated and sporulated cohorts).  Oocysts were thawed and ground with a Teflon pestle. Homogenates werre mixed with chloroform and centrifuged at 12,000 g for 15 minutes at 4°C. The upper aqueous phase was transferred to new tubes and mixed with isopropanol to precipitate total RNA. Total RNA was then washed with 75% ethanol and dried before resuspending in molecular grade DEPC-treated water. Total RNA was quantified using a ThermoFisher Scientific Qubit 3.0 fluorometer and quality was assessed using an Agilent Bioanalyzer 2100 (with RNA 6000 nano kit) and Agilent Tapestation 4150 (with High Sensitivity Screen Tape and reagents). RNA samples were frozen at -80°C. Prior to RNA-Seq, DNA was removed from total RNA with an Invitrogen Turbo DNA-free kit. Libraries were produced from ~200 ng of total RNA from three techincal replicates per cohort using an Illumina stranded mRNA Prep Kit (ligation). For some libraries, excess Illumina adapter was removed by gel electrophoresis size selection using a Blue Pippin instrument. Bioanalyzer and Tapestation analysis determined the average library size of cohorts ranged 324-379 bp. Libraries were sequenced on a MiSeq using a 600 cycle MiSeq Reagent kit at 15 pM loading concentration.