Nutrient Limitation mimics artemisinin tolerance in malaria
QC, Library Preparation with PolyA Selection and HiSeq Sequencing. The RNA samples received were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked using TapeStation (Agilent Technologies, Palo Alto, CA,USA). The RNA sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturers instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered onto 1 flowcell lane. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturers instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification.