GSM1520194: Pf_troph_un_rep1; Plasmodium falciparum; RNA-Seq
Parasites 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. Genomic DNA was removed from total RNA using a Turbo DNA freeTM kit (Ambion, Applied Biosystems). 0.7-4 μg of total RNA from each treatment condition was converted to a 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.
GEO Accession
GSM1520194