Illumina HiSeq 2500 paired end sequencing; RNA-seq of Plasmodium falciparum for comparative analysis of clinical isolates with severe and uncomplicated malaria.
RNA-seq of Plasmodium falciparum for comparative analysis of clinical isolates with severe and uncomplicated malaria.
Informed consent was obtained from patients or their guardians prior to collection of venous blood from patients presenting at hospitals in Timika, Papua, Indonesia who were diagnosed with either uncomplicated or severe malaria. The blood was passed twice over CF11 cellulose columns and the purified red blood cells including  malaria parasite infected red blood cells were collected in PBS, centrifuged and dissolved in ten cell pellet volumes of trizol, incubated at 37 degrees for 5 minutes and then stored at -80 degrees C. CF11 cellulose purified, infected, red blood cells dissolved in trizol and stored frozen were thawed at 37 degrees C, 0.2 x volumes of chloroform was added and samples mixed. incubated 3 min at room temperature, then centrifuged 9,000g for 30 min at 4 degrees. The supernatant was mixed with an equal volume of 70% ethanol and then applied to an RNeasy mini column (QIAGEN). Once all sample had been loaded onto the column using a vacuum manifold 0.7 ml of QIAGEN buffer RW1 was passed through the column, then 2 x 0.5 ml washes with QIAGEN buffer RPE then the column was spun for 1 min and then RNA was eluted from the dried column into a fresh tube using 2 x 0.05 ml aliquots of RNAse free water. RNA was DNased with QIAGEN DNase I in solution as per manufacturer's instructions, removal of gDNA was confirmed by Q-PCR prior to performing the RNA cleanup/DNase I removal protocol with a QIAGEN RNeasy column as per the manufacturer's instructions, RNA was eluted in 2 x 0.05 ml aliquots of RNase free water. Whole RNA integrity was confirmed using a BioRad experion and high sensitivity RNA chips. High quality samples were then depleted of hemoglobin RNA using the Ambion human Globin clear kit as per the manufacturer's instructions except that after capture of globin RNA with streptavidin magnetic beads the remnant supernatant was ethanol precipitated with 3M Na acetate pH5.5 and 0.15 mg/ml linear acrylamide at -20 degrees overnight then washed with cold 70% ethanol and dissolved in 0.05 ml RNase free water. mRNA was enriched using NEBnext Poly(A) mRNA magnetic isolation module but with elution into NEBnext Ultra Directional RNA Library Prep Kit first strand buffer for 10 min at 94 degrees. Library preparation was as per the manufacturer's instructions except that size selection was performed using Ampure XP beads at bead buffer concentrations of 0.5x and 0.8x. Library amplification was performed using 1U Kapa HiFi in 5x HiFi buffer containing TMAC, 0.3 mM each dNTP, 0.5 micromolar each primer, 3 microlitres USER enzyme, incubated at 37 degrees for 15 min, 1 min at 98 degrees then 15 cycles of (10 sec at 98 degrees and 1 min at 65 degrees), then 5 min at 65 degrees. Libraries were purified using 0.8x Ampure XP beads.
Experimental Factor: phenotype
uncomplicated malaria