title: schizont_rep2_RNA
design: RNA in the aqueous phase of cells dissolved in TRIzol reagent (Life Technologies, 15596018) was diluted in 80% ethanol and purified using RNeasy Mini columns (Qiagen, cat 74104). DNA was digested with the RNase-Free DNase Set (Qiagen, cat 79254) until qPCR of the DNased RNA gave a Ct greater than 34 for the P. falciparum hsp70 gene. Total RNA (2.5 g) was depleted of hemoglobin mRNA using the GLOBINclear Kit (Life Technologies, AM1980) and mRNA then purified using magnetic oligo d(T) beads (NEBNext Poly(A) mRNA Magnetic Isolation Module, New England BioLabs, E7490S). Indexed mRNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7420S). The PCR amplification step was performed using 1 unit of high fidelity KAPA HiFi DNA polymerase (KAPA biosystems, KK2101), 0.3 mM dNTPs, 0.5 M NEBNext Universal PCR Primers for Illumina and 0.5 M NEBNext Index Primer for Illumina (NEBNext Multiplex Oligos for Illumina Index Primers Set 1 (E7335) and Set 2 (E7500)) in 1 x KAPA HiFi buffer containing tetramethylammonium chloride for 12 PCR cycles.  The PCR conditions were 98C for 1 minute for the initial denaturation followed by 12 cycles of 98C for 10 seconds and 65C for 1 minute and a final extension at 65C for 5 minutes. Library clean-up was performed using 0.8 x AMPure beads and RNA libraries were eluted with nuclease free water. The libraries were sequenced on a HiSeq2500 (Illumina) generating126 bp paired-end reads.