Scriptseq v2 kit

For each replicate, approximately 5 million treated or untreated THP-1 cells were collected and washed with 1× ice cold PBS. RNA was extracted using the Dynabeads mRNA Direct kit according to manufacturer directions. Sequencing libraries were prepared using the Epicenter ScriptSeq V2 kit according to manufacturer supplied protocol. The resulting libraries were quantified by Qubit, mixed in equal concentrations, and assayed by Bioanalyzer prior to sequencing using Illumina HiSeq 2500 technology.

RNA processing and sequencing has been previously described (Tran et al., 2016 (link)). Briefly, total RNA was extracted from whole blood and depleted of ribosomal and globin RNA prior to amplification using the ScriptSeq Complete Gold Kit (Illumina, San Diego, CA). Directional RNA-seq libraries were prepared using the ScriptSeq v2 kit (Epicentre, Madison, WI). Sequencing of 2×100 bp paired-end reads was performed on a HiSeq 2000 using V3 reagents (Illumina). The Illumina sequences were trimmed of bases with a Phred quality score <15 and any contaminating adapters used in cDNA and sequencing library preparation. Only paired-end reads which survived trimming and were ≥ 60 bases in length were mapped to the human (GRCh37, version 17, Ensembl 72) and P. falciparum 3D7 (version 3 annotation, Dec 2012) genomes in parallel using TopHat2 (Kim et al., 2013 (link)). Transcript abundance was determined by Cufflinks (Trapnell et al., 2012 (link)).