Solid 5500 system

The high-throughput sequencing of four samples (two healthy controls and two patients, P1 and P3) was performed using single-end, 150 nucleotide reads from the 5500 SOLiD System (Life Technologies). All quality color-space reads were cut to 50 nucleotides and mapped against the NCBI Build 37/hg19 genome using LifeScope Genome Analysis Software v2.5.1 with default RNA-Seq parameters (Life Technologies). Expression values of each gene in TPM (transcripts per million) were determined and statistical analysis performed in the RNA-Seq workflow of Partek Genomics Suite (v6.6, St. Louis, MO, USA). Gene Ontology and Function analysis were performed using the PANTHER Classification System (www.pantherdb.org/) [15 (link), 16 (link)] and Ingenuity® Pathway Analysis (IPA) software (www.ingenuity.com), respectively.

An Illumina RNA amplification kit (Thermo-Fisher) was used in order to amplify the small amount of RNA extracted from the cells separated by means of cytometry. Sequencing was performed by means of the 5500 SOLiD system (Life Technologies, Madrid, Spain). Seventy-eight million reads per strain were obtained, and short sequences (75 bp) were assembled and aligned using a reference S. cerevisiae strain (Saccer2). The values of FPKM (Fragments Per Kilobase of exon per Million fragments mapped) were transformed for the statistical test as log2 (FPKM1/FPKM2) and uploaded on Cuffdiff (Cufflinks V1.3.0, http://cufflinks.cbcb.umd.edu/), where FPKM1 stands for the dominant strain data and FPKM2 stands for the non-dominant strain data. The FPKM parameter was used for pair end RNA-Seq experiments, where fragments were sequenced from both ends, and two reads were performed for each fragment. The Q value, which represents the significance value of the statistical analysis, performed through the use of a cufflink platform, was calculated, and significant transcriptome differences were identified. A functional analysis was performed to search for any significantly overrepresented gene ontologies (GO) in a set of genes, using the Genemania functional network predictive online tool30 (link).

For the ookinete culture, six mice were pre-treated with phenylhydrazine and then infected with P. berghei expressing GFP-fused AP2-O. Sulfadiazine was added to their drinking water (10 mg/L) in order to deplete asexual blood-stage parasites. When the exflagellation of the male gametes reached approximately 300 per 10⁵ red blood cells, the blood was harvested and cultured in an ookinete culture medium for 16 h, and then fixed with 1% paraformaldehyde. Erythrocytes in the fixed culture were removed by lysis in 0.84% NH₄Cl, and the remaining ookinetes were subjected to ChIP. ChIP was performed using the ChIP Assay Kit (Millipore) according to the manufacturer’s protocol. Briefly, samples in the lysis buffer were sonicated with a Bioruptor (Tosho Denki, Yokohama, Japan) until chromatin DNA was fragmented to 300–500-bp in size for sequencing with an Illumina Genome Analyzer and to 150-bp for sequencing with a SOLiD 5500 system (Life Technologies). Immunoprecipitation (IP) was performed with anti-GFP antibodies, and the harvested DNA fragments were subjected to sequencing. Input DNAs were obtained from the chromatin without IP. Anti-GFP antibodies used for ChIP were purchased from Clontech and Abcam.