GSM6636929: RNA-seq of Plasmodium blood stages: SZ Rep1; Plasmodium falciparum; RNA-Seq
RNA was isolated from merozoites and schizonts stored in TRI reagent using Zymo's Direct-zol RNA microprep kit according to manufacturer's instructions with DNase treatment. As an extra safe-guard step, isolated RNA was treated with DNase I (Invitrogen #AM1906) using the manufacturer's protocol for rigorous DNase treatment. RNA quality was determined by Bioanalyzer, and its concentration determined by Qubit. RNA samples with an RNA integrity number > 7 were used for library preparation. High quality, strand-specific libraries for RNA-seq were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760S) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) according to manufacturer's instructions with the following modifications. Adaptor-ligated DNA was enriched by PCR with i7 index primers and i5 universal primer (NEBNext multiplex oligos for Illumina index primers set 1 and 2, NEB #E7335 and #E7500) using KAPA HiFi hot start ready mix (Roche # 07958919001) with the following program: initial denaturation at 98°C for 45 sec.; 9 cycles of (98°C for 15 sec., 55°C for 30 sec., 62°C for 30 sec.); and final extension at 62°C for 5 min. Libraries were purified using 0.9 volumes sparQ beads (QuantaBio #95196-060) according to manufacturer's instructions and sequenced with the Illumina NextSeq500 to generate 75 bp paired end reads.