GSM8942329: PF3D7, TP2, WR+, RNAseq; Plasmodium falciparum; RNA-Seq
Following standard manufacturer's instructions, total RNA was extracted using Ribozol, purified and depleted of gDNA by two rounds of purification using the RNeasy Plus Mini Kit and polyA-selected using the Oligotex mRNA Mini Kit. Subsequently RNA was fragmented by alkaline hydrolysis for 1 min 45 sec at 85°C and precipitated with 1 volume of isopropanol, 1/5th volume of 5M ammonium acetate and 2.5 uL of linear acrylamide carrier. Strand-specific RNA-seq was performed. In short, first-strand cDNA synthesis was performed with AT-corrected random N9 primers (76% AT) in the presence of 0.2 µg Actinomycin D (Thermo Fisher Scientific). Second-strand synthesis was performed by replacing dTTPs with dUTPs to preserve the strand-specific information. The synthetized double-stranded cDNAs were end-repaired, extended with 3' A-overhangs and ligated to NextFlex adapters. dUTP-containing second strand DNA was degraded using USER enzyme and libraries were subsequently amplified by PCR (98°C for 2 min; 4 cycles of 98°C for 20 sec, 62°C for 3 min; 62°C for 5 min) using KAPA HiFi HotStart ready mix and NEXTflex primer mix. The resulting amplicons were size-selected for 300-400 bp fragments using 2% E-Gel Size Select agarose gels and amplified for another 12 cycles using the same PCR conditions. Library was purified using Agencourt AMPure XP beads in a 1:1 library:beads ratio.