Protocols: P. falciparum cell culture was performed as described previously (Brancucci, 2015; Trager, 1976). RPMI-1640 culture media were supplemented with 25 mM HEPES, 100 μM hypoxanthine, 24 mM sodium bicarbonate, and gentamicin (all from Sigma-Aldrich). Media containing serum were generated by additional supplementation of 10% O+ human serum (The Interstate Companies). Minimal fatty acid (mFA) media were generated by additional supplementation of 0.39% fatty acid-free BSA, 30 μM oleic acid, and 30 μM palmitic acid (all from Sigma-Aldrich). To generate serum-free media supplemented with 20 μM LysoPC, LysoPC (Avanti Polar Lipids) was dissolved in ethanol and dried on culture dishes prior to culture addition. All cell cultures were maintained in a 5% CO2, 1% O2 and 94 % N2 gas mixture (Med-Tech Gases). Experiments used a clone of the Pf2004/164TdTomato P. falciparum line17, which requires addition of 4 nM WR99210 (Jacobus Pharmaceuticals) to select for stable maintenance of episomes. Additional experiments were performed with strain Dd2, which is a clone derived from W2-MEF which itself is derived from W2'82 (Indochina III/CDC) (Oduola, 1988; Wellems, 1990). Per time point 1mL PCV at ~3% parasitaemia were treated has fallow. Parasites were isolate by saponin treatment (0.01%). RNA extraction was performed with RNeasy plus kit from Qiagen, parasite pellets were resuspended in 350 ¬µl of lysis buffer and stored at -80 degrees C utile extraction. Up to 10 µg of RNA was treated with DNAse Turbo kit (Ambion) prior reverse transcription or RNA seq. RNA quality was check with the Agilent bioanalyzer. 150 ng total RNA of each sample was used for library preparation. RNA quality was check with the Agilent bioanalyzer. Stranded libraries were prepared by using TruSeq Stranded mRNA kit (Illumina) using manufacturer instructions with 13 cycles of PCR. Library quantification was conducted by Qubit measurements using the HS DNA Kit.
ENA-FIRST-PUBLIC
2022-08-31
ENA-LAST-UPDATE
2022-08-31
External Id
SAMEA14277667
INSDC center alias
Institute of Infection, Immunity and Inflammation, University of Glasgow
INSDC center name
Institute of Infection, Immunity and Inflammation, University of Glasgow
INSDC first public
2022-08-31T00:13:23Z
INSDC last update
2022-08-31T00:13:23Z
INSDC status
public
Submitter Id
E-MTAB-11679:97-koM40_S18_R2_001
broker name
ArrayExpress
common name
malaria parasite P. falciparum
developmental stage
IDC
genotype
Ben knockout
growth condition
minimum Fatty Acid medium
organism part
whole organism
sample name
E-MTAB-11679:97-koM40_S18_R2_001
strain
Pf2004