strain: 3D7-A condition: LF parasites from selection 2 in LFCM condition time point: 32-37hpi estimated age (hpi): 32.3
Treatment protocol
3D7-A-Ctrl parasites were always maintained in 0,5% AlbuMAX II medium. The 3D7-A-LF parasites were divided into two conditions after the Percoll synchronization: LF0 (3D7-A-LF parasites always maintained in 0,5% BSA medium) and LFCM (3D7-A-LF parasites switched to 0,5% AlbuMAX II medium).
Growth protocol
Parasites were cultured in B+ erythrocytes at a 3 % haematocrit under standard culture conditions in RPMI-based supplemented with either 0,5% AlbuMAX II (3D7-A-Ctrl parasites) or 0,5% BSA (3D7-A-LF parasites) human serum, in a 5% CO2, 3% O2, balance N2 atmosphere at 35ºC. Tight synchronization (5 h age window) was achieved by Percoll purification followed by sorbitol treatment 5 h later.
Extracted molecule
total RNA
Extraction protocol
RNA was purified using the TRIzol method following the manufacturer instructions.
Label
Cy5
Label protocol
Test sample, Cy5; reference pool, Cy3. As previously described (Painter, H. et al., 2013. PMID: 22990780)
strain: 3D7-A time point: 3 samples of synchronized cultures covering the whole asexual cycle
Treatment protocol
3D7-A-Ctrl parasites were always maintained in 0,5% AlbuMAX II medium. The 3D7-A-LF parasites were divided into two conditions after the Percoll synchronization: LF0 (3D7-A-LF parasites always maintained in 0,5% BSA medium) and LFCM (3D7-A-LF parasites switched to 0,5% AlbuMAX II medium).
Growth protocol
Parasites were cultured in B+ erythrocytes at a 3 % haematocrit under standard culture conditions in RPMI-based supplemented with either 0,5% AlbuMAX II (3D7-A-Ctrl parasites) or 0,5% BSA (3D7-A-LF parasites) human serum, in a 5% CO2, 3% O2, balance N2 atmosphere at 35ºC. Tight synchronization (5 h age window) was achieved by Percoll purification followed by sorbitol treatment 5 h later.
Extracted molecule
total RNA
Extraction protocol
RNA was purified using the TRIzol method following the manufacturer instructions.
Label
Cy3
Label protocol
Test sample, Cy5; reference pool, Cy3. As previously described (Painter, H. et al., 2013. PMID: 22990780)
Hybridization protocol
Hybridization performed as previously described (Painter, H. et al., 2013. PMID: 22990780).
Scan protocol
Microarrays images were obtained using the Agilent G2505C Microarray Scanner.
Data processing
Initial data processing of microarray data, including normalization, was performed using the GE2_1105_Oct12_ACC_2 extraction protocol in Agilent Feature Extraction 11.5.1.1 software. The next steps of the analysis were performed using Bioconductor in an R environment (R version 3.5.3). For each individual microarray, we calculated Cy3 and Cy5 background signal as the median of the 100 lowest signal probes for each channel. Probes with both Cy3 and Cy5 signals below three times the array background were excluded (set to NA). Gene level log2(Cy5/Cy3) values and statistical estimation of parasite age were computed as described (Rovira-Graells et al., 2012. PMID: 22415456). Genes with missing values or genes that in all samples had expression values within the lowest 20th percentile of intensity (Cy5 channel) were excluded from downstream analysis, including identification of differentially expressed genes, because differences in genes expressed at near-background levels are of low confidence. To account for the progression delay induced by HS exposure, which can be a major confounder in the downstream analysis, we estimated the age (hours post-invasion, hpi) of each microarray sample by calculating the most likely time post-invasion along the asexual cycle compared to the HB3 transcriptome as reference, using a statistical likelihood method as previously described [Lemieux et al., 2009, PMID: 19376968]. The file processed_data.txt includes all normalized data, probe name, feature number, gene ID and whether or not the probe was excluded from the analysis.
