title: GSM4805533: Eik2_F12_ko_EXPI_TP1; Plasmodium falciparum; RNA-Seq
library_construction_protocol: Complete RNA was isolated from the samples using Trizol/chloroform extraction followed by isopropanol precipitation and its concentration and integrity was verified using Agilent Bioanalyzer (RNA 6000 Nano kit) and NanoDrop 1000 spectrophotometer. First strand cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System and a 1:1 mix of Oligo(dT) and random primers (Invitrogen). The DNA-RNA hybrids were purified using Agencourt RNACleanXP beads (Beckman Coulter) and the second cDNA strand was synthetized using a 10 mM dUTP nucleotide mix, DNA Polymerase I (Invitrogen) and RNAseH (NEB) for 2.5 h at 16°C. The long cDNA fragments were purified and fragmented using a Covaris S220 system (duty cycles = 20, intensity = 5, cycles/burst = 200, time = 30s). The ~200 bp long fragments were end-repaired, dA-tailed and ligated to “PCR-free” adapters (Kozarewa et al., 2009) with index tags using NEBNext according to the manufacturer's instructions. Excess adapters were removed by two rounds of clean-up with 1 volume of Agencourt AMPure XP beads. Final libraries were eluted in 30 μl water, quality-controlled using Agilent Bioanalyzer (High Sensitivity DNA chip) digested with USER enzyme (NEB) and quantified by qPCR. 