title: GSM2127893: Pf_DCJ_H33Ty1_T30_dirRNAseq; Plasmodium falciparum; RNA-Seq
library_construction_protocol: directional RNA-seq protocol to extract and prepare the material: Parasite cultures used for RNA extraction were immediately placed and processed on ice (4°C).  The preparation of strand-specific RNA-seq libraries was performed as in (Kensche et al., 2015). In short, after total RNA isolation (with on-column DNase treatment) and oligo-dT-selection (Hoeijmakers et al., 2013a) PolyA-selected RNA was further treated with DNase (TURBO DNase, Ambion) to remove remaining genomic DNA.  Subsequently, 1.2µg PolyA-selected total RNA equivalent (see (Hoeijmakers et al., 2013a) for details) for 10hpi and 2µg for 20hpi, 30hpi and 40hpi were fragmented by means of alkaline hydrolysis (5x fragmentation buffer: 200mM Tris acetate pH 8.2, 500mM potassium acetate, 150mM magnesium acetate) for 2min at 85°C in 250µl volume. RNA was precipitated overnight and checked for genomic DNA contamination using qPCR, followed by - if needed - another DNase treatment and RNA precipitation for clean-up. Remaining material was processed for strand-specific RNA-seq as in (Kensche et al., 2015). To prevent unwanted DNA-dependent second strand cDNA synthesis - a major source of artificial antisense ‘transcripts’ (Perocchi et al., 2007) - 0.2µg Actinomycin D was included into the first strand synthesis reaction, which was followed by a 15 min 70 °C enzyme deactivation step and QIAquick MinElute purification (Qiagen). During second strand synthesis replacement of dTTP with dUTPs resulted in incorporation of U-bases into the second cDNA strand. Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification 2ng double stranded cDNA were used for each sequencing library which were prepared as described in (Hoeijmakers et al., 2011) and (Kensche et al., 2015) with a few adjustments: after adaptor ligation samples were treated with USER enzyme (New England Biolabs) for 15min at 37°C that resulted in dUTP-dependent second strand-specific degradation, followed by a first amplification cycle (4 cycles: 98°C for 2min; 4 cycles of 98°C for 20sec, 62°C for 3min; 62°C for 5 min), using 2x KAPA HiFi HotStart ready-mix (KAPA Biosystems) and NextFlex primers, size-selection for 300-400bp fragments using 2% E-Gel Size Select Agarose Gels (Thermo Fisher Scientific), a second amplification cycle (9 cycles) and Agencourt AMPure XP Beads purification  (1:1 library beads ratio).
attributes.tag: GEO Accession
attributes.value: GSM2127893