|
| Status |
Public on Jun 01, 2023 |
| Title |
PB-58 22hpi rep2 |
| Sample type |
RNA |
| |
|
| Source name |
PB58
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: Isogenic mutant of NF54
time point (hours post invasion): 22
|
| Treatment protocol |
Culture were maintained under standard culture conditions.
|
| Growth protocol |
The parasite strains NF54 and PB58 were maintained in identical standard culture conditions in human red blood cells (Indiana Regional Blood Center, Indianapolis, Indiana) suspended in complete medium (CM) containing RPMI 1640 with L-glutamine (Invitrogen Corp.), 50 mg/L hypoxanthine (Sigma-Aldrich), 25 mM HEPES (Cal Biochem), 0.5% Albumax II (Invitrogen Corp.), 10 mg/L gentamicin (Invitrogen Corp.) and 0.225% NaHCO3 (Biosource) at 5% hematocrit. Cultures were grown separately in sealed flasks at 37˚C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Three replicated cultures were thawed and triple sorbitol synchronized. The time points collected were 6 (n=3), 26 (n=5), and 38 (n=3) hours post invasion (hpi).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using TriZol reagent (Invitrogen, Carlsbad, CA) and the quality and quantity determined by NanoDrop (NanoDrop Technologies). 300ng of RNA was used as starting material for cDNA synthesis using the Sigma WTA2 whole transcriptome amplification kit (Sigma Aldrich, St Louis, MO).
|
| Label |
Cy3
|
| Label protocol |
1µg of cDNA was labeled with Cy3 dye using 65% AT rich pre-labeled random hexamers as primers for cDNA synthesis by Klenow fragment of DNA polymerase I.
|
| |
|
| Hybridization protocol |
Cy3 labeled cDNA was allowed to hybridize for 17 h to a custom Agilent microarray (GPL22280) at 65°C at 10 rpm
|
| Scan protocol |
The microarray image was taken using a 2 μM scanner and probe intensity values were obtained using Agilent Feature Extraction software.
|
| Data processing |
Probe intensities for all samples were quantile normalized. Samples were visualized using Principle Component Analysis. Combat and SVA packages in R were used to remove batch effects due to RNA processing, cDNA creation, cDNA labeling and microarray hybridization. Transcript expression levels were summarized for each gene by averaging the batch corrected processed signal intensity of all the probes across its exons.
|
| |
|
| Submission date |
Sep 05, 2018 |
| Last update date |
Jun 01, 2023 |
| Contact name |
Michael T Ferdig |
| E-mail(s) |
ferdig.1@nd.edu
|
| Organization name |
University of Notre Dame
|
| Department |
Biological Sciences
|
| Lab |
Ferdig lab
|
| Street address |
100 Galvin Life Sciences Center
|
| City |
Notre Dame |
| State/province |
IN |
| ZIP/Postal code |
46556 |
| Country |
USA |
| |
|
| Platform ID |
GPL22280 |
| Series (1) |
| GSE119516 |
Plasmodium falciparum intraerythrocytic stage transcription time course for NF54 and isogenic mutant line with altered K13 expression II. |
|
