Directional RNA-seq of Plasmodium falciparum

To select for polyA+ RNA (mRNA), P. falciparum-derived total RNA was bound to magnetic oligo-d(T) beads and purified. Full-length mRNA was primed with oligo d(T) primers, and reverse transcribed using Superscript II (Life). Second strand synthesis used deoxynucleotides dATP, dUTP, dGTP and dCTP to encode directional information. The resulting cDNA was sheared using a Covaris AFA sonicator, and consecutive library preparation steps (dA-tailing, end repair and adapter ligation) were performed in the same well using a “with-bead” approach (reagents from NEB, equivalent to kit E6040). To avoid amplification bias (as described [19 (link)]), we used barcoded sequencing adaptors (Bioo Scientific), followed by 2 rounds of cleanup with AmpureXP beads (Beckmann Coulter) into EB buffer. To produce directional libraries, second strand cDNA was digested using USER enzyme mix (NEB). Prior to sequencing on an Illumina HiSeq2000 (100 bp paired-end), qPCR was used to quantify all libraries. The reads were mapped to version 3 of the 3D7 reference genome [20 (link)] using directional parameters in TopHat2 [73 (link)] and a maximum intron size of 5000 nt.