title: GSM6372419: KD, S3; Plasmodium falciparum; ssRNA-seq
library_construction_protocol: Samples for RNA-seq analyses were taken at 12, 24, and 36 hpi from highly synchronized cultures. For RNA analyses, erythrocyte pellets were rapidly lysed in 10× volumes of pre-warmed TRIzol (ThermoFisher, Waltham, USA) and stored at -80°C until RNA purification. The experiment was repeated at 4-week intervals to obtain three RNA samples as biological replicates. RNA purification and DNase treatment of the samples were performed as described previously (91). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform with paired-end 150 bp read chemistry. Raw reads in fastq format were firstly processed through in-house Perl scripts. The UMI (Unique Molecular Identifiers) was extracted by UMI-tools v1.0.0 (92). All the downstream analyses were based on high-quality UMI reads (92). Strand-specific RNA-seq paired-end reads were mapped onto the P. falciparum 3D7 genome (assembly GCA_000002765) using Hisat2 v2.0.4 (93). UMI-tools v1.0.0 were used to deduplicate reads based on the mapping coordinates and the UMI attached to the reads (92). HTSeq v0.9.1 was used to count the reads mapped to each gene. The FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of genes between the WT and NIF4iKD lines (three biological replicates per condition) was performed using the DESeq R package (1.18.0). The resulting P-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed. GO enrichment analysis of differentially expressed genes was implemented using the GOseq R package, in which gene length bias was corrected (94). GO terms with corrected P value less than 0.05 were considered significantly enriched by differentially expressed genes. RNA libraries were prepared for sequencing using standard illumina protocols