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| Status |
Public on Sep 17, 2022 |
| Title |
Transcriptome of P. falciparum Dd2 untreated (for CQ Dd2 Bioreplicate 2 experiments). Technical replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
In vitro culture, asexual blood stage, laboratory strain Dd2, treated with antimalarial
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: Dd2
treatmant: CQ Control
|
| Treatment protocol |
Parasite culture was divided into 10 well in two 6-well plates with each well containing 6 mL of culture at 3% parasitaemia and 2% hematocrit and treated with 4-fold dilution series of CQ (10 µM top concentration), MK4815 (20 µM top concentration), MFQ (10 µM top concentration), primaquine (50 µM), and an equivalent volume of DMSO at 0.1% concentration and incubated for 6 hours at 37°C with 5% CO2, 3% O2, and 92% N2. Following incubation, the samples were harvested and centrifuged at 2,500 rpm for 5 minutes. Supernatant was removed and 1 mL of trizol was added the the pellets
|
| Growth protocol |
P. falciparum strain 3D7 and Dd2 strains was obtained from BEI Resources and cultured under standard conditions in RPMI1640 media. Prior to drug treatment, parasites were double-synchronized with 5% sorbitol solution to achieve a synchrony of +/- 6 hours and cultured under constant agitation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions and as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211.
|
| Label |
Cy5
|
| Label protocol |
Reverse transcription and cDNA synthesis were done following modifed Smart-Seq2 protocol (Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181 (2014)). Synthesis of aminoallyl-coupled cDNA and labeling of samples with Cy fluorophore (GE Healthcare) were carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211.
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| |
|
| Channel 2 |
| Source name |
In vitro culture, asexual blood stage, laboratory strain 3D7, mixture of equal amounts of RNA harvested every 6 hours over the 48-hour parasite's intraerythrocytic life cycle
|
| Organism |
Plasmodium falciparum |
| Characteristics |
condition: RNA reference pool
|
| Treatment protocol |
Parasite culture was divided into 10 well in two 6-well plates with each well containing 6 mL of culture at 3% parasitaemia and 2% hematocrit and treated with 4-fold dilution series of CQ (10 µM top concentration), MK4815 (20 µM top concentration), MFQ (10 µM top concentration), primaquine (50 µM), and an equivalent volume of DMSO at 0.1% concentration and incubated for 6 hours at 37°C with 5% CO2, 3% O2, and 92% N2. Following incubation, the samples were harvested and centrifuged at 2,500 rpm for 5 minutes. Supernatant was removed and 1 mL of trizol was added the the pellets
|
| Growth protocol |
P. falciparum strain 3D7 and Dd2 strains was obtained from BEI Resources and cultured under standard conditions in RPMI1640 media. Prior to drug treatment, parasites were double-synchronized with 5% sorbitol solution to achieve a synchrony of +/- 6 hours and cultured under constant agitation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions and as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211.
|
| Label |
Cy3
|
| Label protocol |
Reverse transcription and cDNA synthesis were done following modifed Smart-Seq2 protocol (Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181 (2014)). Synthesis of aminoallyl-coupled cDNA and labeling of samples with Cy fluorophore (GE Healthcare) were carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of labelled sample and reference cDNA were hybridized on the microarray chip using the Agilent hybridization system. Hybridizations were performed for 20 hours at 70°C in a rotating hybridization oven at 10rpm as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923:
|
| Scan protocol |
Microarray scanning was done using PowerScanner (Tecan) at 10uM resolution and with autogain PMT adjustments for both channels. Data was acquired using GenePix Pro v6.0 software (Axon Instruments) as described in Mok, S., M. Imwong et al (2011) Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription. BMC Genomics 12: 391.
|
| Data processing |
Features with flag>0 and with median foreground intensity greater than 1.5 fold median background intensity for either channel passed quality control and were accepted.
Local background correction using normexp, followed by lowess normalization across all features within array and quantile normalization between arrays were carried out to generate the normalized Log2 gene expression ratios.
Background corrected and normalized log2 transformed ratios (Cy5/Cy3) of the RNA sample/3D7 RNA reference pool.
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| |
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| Submission date |
Mar 16, 2022 |
| Last update date |
Sep 18, 2022 |
| Contact name |
Zbynek Bozdech |
| E-mail(s) |
zbozdech@ntu.edu.sg
|
| Phone |
63162925
|
| Organization name |
Nanyang Technological University
|
| Street address |
50 Nanyang Avenue
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
639798 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL18893 |
| Series (1) |
| GSE198746 |
Dose Response Transcriptional Profiling of Plasmodium falciparum 3D7 and Dd2 strains treated with chloroquine (CQ), MK-4815, and mefloquine (MFQ) |
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