title: GSM5385948: E. acervulina t12-B; Eimeria acervulina; RNA-Seq
library_construction_protocol: Total RNA was extracted from frozen oocyst Trizol pellets using two methods. Oocysts were thawed and ground with a Teflon pestle. Homogenates were vortexed with 0.5 mm beads, mixed with chloroform, and centrifuged at 12,000 g for 15 minutes at 4°C. The organic phase was transferred to new tubes and mixed with 70% ethanol. Total RNA was then isolated using a RNeasy kit  following manufacturer's instructions. The second method included these modifications: Organic layers from chloroform extraction were mixed with isopropanol and incubated for 10 min at room temperature. Tubes were centrifuged at 12,000 g for 10 min at 4°C. Pellets were resuspended in 1 ml of 75% ethanol, vortexed, and centrifuged at 7,500 g for 5 min at 4°C. Isolated RNA was resuspended in RNase-free water and incubated at 65°C for 15 min. Total RNA was quantified using a Qubit 3.0 fluorometer and quality was assessed using a Bioanalyzer 2100. RNA samples were frozen at -80°C. Prior to RNA-Seq, DNA was removed from total RNA with a Turbo DNA-free kit. Libraries were produced from 200 ng of total RNA from 2-3 biological replicates using an Illumina TruSeq Stranded mRNA kit. cDNA libraries were selected for fragment sizes of between 120-210 bp.