title: Generic sample from Plasmodium berghei
organism: Plasmodium berghei
description: BIOLOGICAL MATERIAL P. berghei Ring, 4 hours post-invasion ; <5% gametocytes contamination ; EXTRACTION PROTOCOL AND LIBRARY CONSTRUCTION Total RNA was extracted using Trizol method. Then extracted total RNA was first cleaned over an RNeasy Minelute collumn with on collumn DNase digestion for a subset of samples (cl15cy1_Ring, cl15cy1_Gametocyte & cl15cy1_Ookinete) or processed directly (cl15cy1_Trophozoite, cl15cy1_Schizont & all replicates) for PolyA-selection using Oligotex mRNA mini Kit (Invitrogen). After PolyA selection, PolyA+ RNA was fragmented using hydrolysis and in-solution DNase treatment was performed for the subset of samples that was not DNase treated yet (cl15cy1_Trophozoite, cl15cy1_Schizont & all replicates). Fragmented RNA was converted to double-stranded cDNA using 76%AT containing random N9 primers (all according to the protocol described in Hoeijmakers WAM, Bartfai R, Stunnenberg HG. Methods in Molecular Biology, 2013). 15-40ng of total RNA was used for sequening library preparation using the LADS protocol (Hoeijmakers WAM, Bartfai R, Francoijs K-J, Stunnenberg HG, Nature Protocols, 2011). Integrity of the total RNA was verified on agarose gel while absence of genomic DNA contamination was verified using a RT-control cDNA synthesis and qPCR. Libraries were sequenced on a Illumina GAIIx machine for 76 cycles using Standard Cluster Generation Kit V4 and V4 Sequencing Reagents.   
attributes.tag: strain
attributes.value: ANKA (1037m1f1m1cl1)
attributes.tag: dev_stage
attributes.value: Ring, 4 hours post-invasion
attributes.tag: sample_type
attributes.value: PolyA+ RNA
attributes.tag: cell_type
attributes.value: asexual intra-erythrocytic development
attributes.tag: culture conditions
attributes.value: Synchronized asexual infections were established in Wistar rats by intravenous injection of purified mature schizonts (time-point 0 hour), resulting in highly synchronous infections of 1-3% parasitemia (Janse and Waters, Parasitol. Today, 1995). 4 hours after injection of schizonts, infected blood (containing rings 0-4 hours post-invasion) was collected, leukocytes were removed and parasites were further cultured at 37°C under standard in vitro conditions (Janse and Waters, Parasitol. Today, 1995). Rings, trophozoites and schizonts were collected 4, 16 and 22 hours after injection of schizonts. Mature gametocyte populations were obtained by treatment of P. berghei infected mice with sulfadiazine and collected from the mice by heart puncture, after which leukocytes were removed and gametocytes were purified using Nycodenz gradient centrifugation (Beetsma, van der Wiel, Sauerwein. Eling, Exp. Parasitol, 1998). Ookinetes were producted from the purified gametocyte cultures by standard in vitro culture for ookinete production at 21C for 22 hours (Janse et al., Parasitology, 1985). ;