title: GridION sequencing; Long read RNA-seq of Plasmodium falciparum 3D7 for annotation of long non-coding RNAs
design: Long read RNA-seq of Plasmodium falciparum 3D7 for annotation of long non-coding RNAs
library_construction_protocol: P. falciparum parasites (3D7 strain) were grown as blood-stage cultures in RPMI media with AlbuMAX® (Gibco) and supplemented with GlutaMax® (Gibco), Gentamicin (Gibco) and HEPES (pH of 7) with O+ human erythrocytes at 3% haematocrit. Cultures were maintained at 37oC in a gaseous environment of 3% CO2, 1% O2 and 96% N2. Parasitemia and stages were monitored using Giemsa staining and microscopy.  RNA was extracted from parasites using Trizol as previously described in Kyes, S., Pinches, R. & Newbold, C. A simple RNA analysis method shows var and rif multigene family expression patterns in Plasmodium falciparum. Molecular and biochemical parasitology 105, 311-315, doi:10.1016/s0166-6851(99)00193-0 (2000). RNA samples were run on the Oxford Nanopore Gridion using the direct RNA-seq protocol, avoiding PCR.