|
| Status |
Public on Jul 31, 2020 |
| Title |
Time 56_PyrkII CLD_Rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
PyrkII CLD Nf54 attB parasites,Time 56, Rep 3
|
| Organism |
Plasmodium falciparum |
| Characteristics |
tissue: Blood-stage PyrkII CLD Nf54 attB P. falciparum parasites cultured in human erythrocytes at 2% hematocrit in media containing 500nM Shield1
time: Time 56
treatment: PyrkII CLD
slide id (array batch): 253723710175
replicate: Rep 3
|
| Treatment protocol |
The CLD-PyrKII parasite line was synchronized via magnetic purification and used to seed a 50mL culture flask at 1% parasitemia and 2% hematocrit. Six hours post synchronization (mid-schizogeny) 500nM Shield1 was added, representing time 0. Media containing 500nM Shield1 was replaced at 16 hours and every 24 hours thereafter.
|
| Growth protocol |
Blood-stage P. falciparum parasites were cultured in human erythrocytes at 2% hematocrit in CMA (Complete Medium with Albumax) containing RPMI 1640 medium with L-glutamine (USBiological Life Sciences), supplemented with 20mM HEPES, 0.2% sodium bicarbonate, 12.5μg/ml hypoxanthine, 0.5μM R-lipoic acid, 5g/L Albumax II (Life Technologies) and 25μg/mL gentamicin. Cultures were maintained in 25cm2 gassed flasks (94% N2, 3% O2, 3% CO2) and incubated at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each data point, RNA was extracted from 100 µL of packed RBC culture with Trizol Reagent and the PureLink Mini Kit (Invitrogen/Thermo Fisher Scientific). 1ml Trizol was added to each sample, followed by high-speed disruption in tubes containing Lysing Matrix D (MP Biomedical) in a FastPrep 120 Instrument at speed 6 for 20 seconds. Homogenates were subsequently processed according to the PureLink Mini Kit manufacturer’s (Invitrogen/Thermo Fisher Scientific) protocol, including on-column DNase treatment.
|
| Label |
Cy3
|
| Label protocol |
From 100 ng of total RNA, Cyanine-3 labeled cRNA was prepared using the Low Input Quick Amp Labeling Kit One-Color (Agilent).
|
| |
|
| Hybridization protocol |
The eight time course samples were hybridized (along with the manufacturer’s RNA spike-in controls) according to standard protocol to the 8 sectors of an Agilent 8x15K platform microarray (AMADID 037237) with separate microarray slides used for each biological replicate (in triplicate for each treatment condition).
|
| Scan protocol |
Arrays were scanned with the Agilent G2600D SureScan microarray scanner using scan protocol AgilentHD_GX_1color. Agilent’s Feature Extraction Software was used to assign grids, provide raw image files per array, and generate QC metric reports from the microarray scan data.
|
| Description |
Gene Expression Data
|
| Data processing |
Txt files from the Feature Extraction software were imported into the Partek Genomics Suite (v7.0; Partek) for detailed analyses of gene expression. Within Partek, the gProcessedSignal was imported, and the intensity values were normalized to the 75th percentile, lower expressed genes were filtered out using a max cutoff of <=0.5, and log transformation base 2.0 was performed.
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| |
|
| Submission date |
Aug 30, 2019 |
| Last update date |
Jul 31, 2020 |
| Contact name |
Anne E. Jedlicka |
| Organization name |
Johns Hopkins University, School of Public Health
|
| Department |
Molecular Microbiology and Immunology
|
| Lab |
Genomic Analysis and Sequencing Core
|
| Street address |
615 N. Wolfe street
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL27130 |
| Series (1) |
| GSE136688 |
The NTP Generating Activity of Pyruvate Kinase II is Critical for Apicoplast Maintenance in Plasmodium falciparum |
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