parental strain: 3D7-B timepoint: 30-35 hpi estimated age (hpi): 30.2
Treatment protocol
No treatment was applied.
Growth protocol
Samples for transcriptomic analysis of A7, E5 and B11 lines were obtain from tightly synchronized cultures (0-5 h window) achieved by Percoll purification of erythrocytes infected with mature stages followed by 5% sorbitol lysis 5 h later. RNA was extracted from samples collected at 10-15, 20-25, 30-35, 37-42, 40-45 and 43-48 h post-invasion (hpi) using the Trizol method. After reverse transcription and labelling, Cy5-labelled samples where hybridized against a Cy3-labelled reference pool. The reference pool consisted of a mixture of equal amounts of cDNA from rings, trophozoites and schizonts from 3D7-A and 3D7-B stocks. Samples were hybridized oncustom Agilent microarrays (AMADID no. 085763) modified from design AMADID no. 037237.
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 and 3D7-B timepoint: mixture of equal amounts of cDNA from rings, trophozoites and schizonts from 3D7-A and 3D7-B
Treatment protocol
No treatment was applied.
Growth protocol
Samples for transcriptomic analysis of A7, E5 and B11 lines were obtain from tightly synchronized cultures (0-5 h window) achieved by Percoll purification of erythrocytes infected with mature stages followed by 5% sorbitol lysis 5 h later. RNA was extracted from samples collected at 10-15, 20-25, 30-35, 37-42, 40-45 and 43-48 h post-invasion (hpi) using the Trizol method. After reverse transcription and labelling, Cy5-labelled samples where hybridized against a Cy3-labelled reference pool. The reference pool consisted of a mixture of equal amounts of cDNA from rings, trophozoites and schizonts from 3D7-A and 3D7-B stocks. Samples were hybridized oncustom Agilent microarrays (AMADID no. 085763) modified from design AMADID no. 037237.
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
Normalized Cy5 and Cy3 values were obtained directly using Agilent Feature Extraction software. The rest of the analysis was performed using mainly the Bioconductor and the tidyverse suite libraries in an R environment (v. 3.6.3). IFor each channel, background signal intensity was calculated as the median signal of the 100 lowest signal probes in each array. Probes with a signal for both channels below three times the background were excluded from further analysis. For each probe, the expression value was defined as the log2(Cy5/Cy3) signal. Probes were collapsed into genes using median polish. For each array, estimated parasite age in hpi was calculated using a previously described maximum likelihood method [Lemieux et al., 2009, PMID: 19376968]. For each gene and subclone, expression plots where generated with gene expression values in the y-axis and estimated hpi in the x-axis. These plots were used to calculate the average fold-change (AFC) for each gene over four overlapping time intervals of half the estimated parasite age difference (in hpi) between the first and last analysis time points. The AFC was calculated for all possible pairwise comparisons among A7, E5 and B11. The maximum AFC (mAFC) was defined as the AFC at the time interval in which it had the highest absolute value. The file processed_data_with_genename.txt includes all normalized data with gene annotations and which probes were excluded from analysis
Patterns of heterochromatin distribution alterations linked to transcriptional changes at Plasmodium falciparum clonally variant gene loci [gene expression]
