title: GSM9131404: TGGT1_321450_6h_IAA_1; Toxoplasma gondii; RNA-Seq
library_construction_protocol: Monolayers were aspirated, rinsed with PBS, and parasites mechanically released by scraping and passage through a 27-gauge needle, followed by filtration through a 5  μm membrane. Parasite suspensions were centrifuged at 1,500 x g for 5  min, and RNA was extracted from parasite pellets by double extraction using TRIzol (Invitrogen). Therefore, the pellet was resuspended in 800 µl TRIzol Reagent (Invitrogen), vortexed, and split into two different tubes to retain one technical replicate. Phase separation was performed in phase lock heavy tubes (Quantabio) by addition of 100  μl chloroform:isoamyl alcohol (24:1, Sigma-Aldrich), vigorous vortexing (1 min), and centrifugation at 16,000 x g for 5  min at 4 °C. The aqueous phase was isolated, and TRIzol extracted again using 400 µl TRIzol. The resulting aqueous phase was supplemented with 1 μl Glycogen (20 mg/ml, Thermo Fisher Scientific) and 500 μl isopropanol, and RNA was precipitated at room temperature for 12 min. Pellets were recovered by centrifugation (12,000 x g, 12  min) at 4 °C, washed twice with 70 % ethanol, air-dried for 10  min at room temperature, and resuspended in 20 μl RNase-free dH2O. RNA-seq libraries were prepared with the Rapid Directional RNA-Seq Kit 2.0 (NEXTFLEX) using Poly(A) Beads 2.0 (NEXTFLEX).