GSM6855939: +aTc TetR-PfGCN::GFP L-Glu rep2; Plasmodium falciparum; RNA-Seq
Parasites of designated developmental stages were harvested after lysis of the RBC membrane with 0.1% (v/v) saponin in PBS. Total RNA was isolated from the purified parasites using Quick-RNA MiniPrep kit (Zymo Research). The RNA was treated with the ezDNaseTM Enzyme (Invitrogen) to remove contaminating genomic DNA. RNA pellets were dissolved in 50 µL RNase-free water, and the RNA integrity was checked using the Agilent Bioanalyzer. RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. RNA was fragmented by heating at 94 °C for 8 min, and cDNA was synthesized using reverse transcriptase with random primers. Afterward, 3' dTMP adapters were ligated to the 3' dAMP library fragments. The quality of the barcoded libraries was validated using the Agilent Bioanalyzer. The DNA 1000 Reagents were used to quantify the library sizes and confirm the absence of primer dimers. Libraries were quantified using a KAPA Universal Library Quantification Kit (Roche), and library concentrations were adjusted for library size. Libraries were sequenced on an Illumina NextSeq 550 in the Rapid Run mode using 150 nt paired end read sequencing .