GSM6434028: DD2_DHA_04; Plasmodium falciparum; RNA-Seq
After 6 hours of DHA or DMSO treatment, we centrifuged the parasite culture mix at 2500rpm for five minutes to harvest the infected erythrocytes. Subsequently, parasites were liberated from host erythrocytes by treatment with 0.015 % (w/v) saponin for five minutes at room temperature. After that, liberated parasites were washed twice with 1x PBS (137mM NaCl, 8mM KCl, 10mM Na2HPO4, 2mM KH2PO4, pH 7.4) to remove any host cell debris and residual drug. The parasites were then pelleted and stored at -80oC in an Eppendorf tube (1.5ml) containing 1mL Trizol reagent (Fisher Scientific, Hampton, NH) before RNA extraction. Sequencing was performed at Single Cell Discoveries, a sequencing service provider located in the Netherlands using an adapted version of the CEL-seq protocol. Total ribonucleic acid (RNA) was extracted from TRIzol-preserved parasites after thawing at room temperature using the standard TRIzol (Invitrogen) protocol and used for library preparation and sequencing. The extracted RNA was dissolved in 100µl RNase-free water and DNA removed by DNase I digestion, after which the purified DNA was eluted into 50µl RNase-free water. Qubit High Sensitivity RNA assay (Thermo Fisher Scientific) was used to quantify the purified RNA, and RNA integrity was determined on an Agilent Bioanalyzer (Agilent Genomics, Waldbronn, Germany). Messenger RNA (mRNA) was processed as described previously, following an adapted version of the single-cell mRNA sequencing protocol of CEL-Seq (Hashimshony et al., 2012, Simmini et al., 2014). In brief, samples were barcoded with CEL-seq primers during reverse transcription and pooled after second strand synthesis. The resulting complementary DNA (cDNA) was amplified with an overnight in vitro transcription reaction. From this amplified RNA, sequencing libraries were prepared with Illumina Truseq small RNA primers. The DNA library was paired end sequenced on an Illumina NextseqTM 500, high output, with a 1x75 bp Illumina kit (R1: 26 cycles, index read: 6 cycles, R2: 60 cycles). Read 1 was used to identify the Illumina library index and CEL-Seq sample barcode. Read 2 was aligned to the Plasmodium falciparum ASM276v2 reference transcriptome using BWA, version 0.7.17-r1188 (Li and Durbin, 2009).