NextSeq 500 sequencing; RNA immunoprecipitation of Plasmodium berghei coupled to RNA-sequencing of GBP2::mCherry parasites.
RNA immunoprecipitation of Plasmodium berghei coupled to RNA-sequencing of GBP2::mCherry parasites.
Protein IP Infected erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The infected erythrocytes were incubated for 18 h in 90% N2, 5% CO2 and 5% O2. Mature schizonts and gametocytes were harvested by Nycodenz density gradient centrifugation, as described previously15. Proteins were then extracted using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Protein IP in transgenic parasites expressing mCherry fused to GBP2 or NAB2 was performed using GFP- or RFP-Trap Agarose and the GFP-Trap-A kit, according to the manufacturer's instructions (Chromotek, Planegg, Germany).   Following protein IP, RNA was isolated using the RIP-Assay Kit (Medical ＆ Biological Laboratories, Tokyo Japan) according to the manufacturer's instructions. RNA from two independent RNA IP assays were prepared to generate cDNA using SMART-Seq V4 Ultra Low Input RNA Kit for Sequencing Kit (Takara, Shiga, Japan).