title: GSM5001788: WT_2_Gamet_30min; Plasmodium berghei; ssRNA-seq
library_construction_protocol: Total RNA was isolated from TRIzol treated samples according to the manufacturer's instructions (Life Technologies). Strand-specific mRNA sequencing was performed of total RNA and using TruSeq stranded mRNA sample prep kit LT (Illumina), as previously described55.  Libraries were sequenced using an Illumina Hiseq 4000 sequencing platform with paired-end 150 bp read chemistry. The quality of the raw reads was assessed using FATSQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Low-quality reads and Illumina adaptor sequences from the read ends were removed using Trimmomatic R56. Processed reads were mapped to the P. beghei ANKA reference genome (release 40 in PlasmoDB- http://www.plasmoddb.org) using Hisat257 (V 2.1.0) with parameter “—rna-strandness FR”. Counts per feature were estimated using FeatureCounts. Raw read counts data were converted to counts per million (cpm) and genes were excluded if they failed to achieve a cpm value of 1 in at least one of the three replicates performed. Library sizes were scale-normalized by the TMM method using EdgeR software59 and further subjected to linear model analysis using the voom function in the limma package. Differential expression analysis was performed using DeSeq.  Genes with a fold-change greater than two and a false discovery rate corrected p-value (Benjamini-Hochberg procedure) < 0.05 were considered to be differentially expressed. Genes with fold change greater than 2 and p-value less than 0.05 were considered as significantly differentially expressed. Stranded RNA-Seq