Pberghei_ANKA_cl15cy1_Ookinete_RNAseq_61WR1AAXX_6
Total RNA was first cleaned over an RNeasy Minelute collumn with on collumn DNase digestion for a subset of samples (cl15cy1_Ring, cl15cy1_Gametocyte and cl15cy1_Ookinete) or processed directly (cl15cy1_Trophozoite, cl15cy1_Schizont and 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 and 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.