title: GSM1680587: VN_seq_f7; Plasmodium falciparum K1; RNA-Seq
library_construction_protocol: Parasites (mature stages, mostly trophozoite and schizonts) were harvested from culture and liberated from host cells by treatment with 0.15% (w/v) saponin for 5 min on ice. The liberated parasites were washed three times with 1xPBS (137 mM NaCl, 8 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to remove host cell debris and drug. Total RNA was extracted from parasites using Trizol reagent (Invitrogen) following the manufacturers’ recommendations. 5' capped mRNA was purified as described in Shaw et al. (2007) Mol Biochem Parasitol 155: 146-155. 1 mg of GST-PfeIF4E SP-sepharose purified protein was bound to 0.2 ml of glutathione-mag beads (Thermo Scientific). 100 micrograms of parasite total RNA was incubated for 15 min on ice with 0.1 ml suspension of the GST-PfeIF4E bound beads. The beads were washed four times with 1 ml 1xPBS and 5' capped mRNA eluted in 0.1% SDS  200 mM NaCl. Eluted mRNA was purified by acidic phenol:chloroform and ethanol precipitation. Genomic DNA was removed from mRNA using a Turbo DNA freeTM kit (Ambion, Applied Biosystems). 800 ng of 5' cap enriched mRNA was used to make unfractionated RNA library (f0). 7.2 μg of 5' cap enriched mRNA was denatured with RNA loading dye (Thermo scientific) and separated in 0.8 % agarose with 1x MOPS gel running buffer. 8 RNA fractions of different sized-RNA were obtained by gel-purification using a ZYMOClean RNA purification kit (ZYMO Research). Approx. 100-400 ng of RNA from each fraction was converted to strand-specific RNA-seq sequencing library using a TruSeq Stranded mRNA LT Sample Prep kit following the manufacturers’ recommendations (Illumina). A different TruSeq adapter was used for each RNA-seq library to allow pooling of samples for Illumina sequencing on the same flow cell. ). The RNA-seq libraries were quantified by QuantIT dsDNA High Sensitivity kit (Invitrogen) and by qPCR using a library quantification kit (Kapa Biosystems). Libraries were pooled in equimolar ratios and applied to a MiSeq flow cell at 12 pM with 1% phiX174 control library (Illumina). Paired-end sequencing was performed on two MiSeq v2 2x150 bp flow cells according to the manufacturers’ recommendations (Illumina). The raw data from the MiSeq instrument were de-multiplexed and fastq files generated for each RNA-seq library.
attributes.tag: GEO Accession
attributes.value: GSM1680587