|
| Status |
Public on Nov 29, 2022 |
| Title |
NF54-T24-Rep-2 |
| Sample type |
SRA |
| |
|
| Source name |
Plasmodium falciparum
|
| Organism |
Plasmodium falciparum NF54 |
| Characteristics |
genotype: wildtype
disrupted gene id: n/a
developmental stage: early trophozoite (T24)
tissue: whole body
|
| Treatment protocol |
Cultures of wildtype NF54 and PB50 (KIC5 mutant) were synchronized at ~2% ring stage with 10% sorbitol solution. After 48 hours of growth, cultures were again synchronized with 10% sorbitol. After 8hrs, cultures were synchronized for a third time with 10% sorbitol to yield highly synchronized cultures. 0hpi was determined (half schizonts and half ring cultures) and samples were collected for RNA extraction and sequencing at 6hpi, 12hpi, 24hpi, 36hpi, and 48hpi.
|
| Growth protocol |
Parasite cultures were maintained according to standard methods with gassing (5% O2 and 5% CO2, nitrogen balanced) and 5% hematocrit (O+ blood; Interstate Blood Bank) in RPMI 1640 medium (Invitrogen) supplemented with 0.5% Albumax II (Invitrogen), 0.25% sodium bicarbonate, and 0.01 mg/ml gentamicin, maintained at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Parasites were separated from red blood cells via incubation with 0.015% saponin at room temperature for 5 min. Cultures were then pelleted and washed three times in cold PBS. Samples were stored at − 80 °C in 1 mL TRIzol reagent (Fisher Scientific, Hampton, NH) until extraction. At RNA extraction, 200 μl of chloroform was added and samples were vortexed vigorously for 15 seconds, followed by incubation at room temperature for up to 5 minutes. Samples were then spun down at 12000×g (10,800 rpm) at 4 °C for 10 minutes and the supernatant discarded. 1 mL of 75% ethanol was added to the pellet, and samples were spun down at 10000×g (9800 rpm) for 5 minutes. The resulting supernatant was discarded and the pellet briefly allowed to dry. The RNA pellet was then dissolved in 20–50 μl of DEPC-treated water while being incubated at 55 °C for 10–15 min.
0.5 μg–1.0 μg of RNA samples were prepped for sequencing using the Illumina TruSeq Stranded mRNA Kit as per kit protocol. Library quantification was measured by qPCR and TapeStation (Agilent Technologies). Sequencing was performed on an Illumina NextSeq V2.5 mid-output 300-cycle.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
control
|
| Data processing |
RNAseq reads from each sample were aligned to the P. falciparum reference genome using HISAT2.
Mapped reads were used to assemble known transcripts from the reference and their abundances were estimated using SAMtools and the abundances were estimated using Feature Counts.
Assembly: P. falciparum reference genome (PlasmoDB version 47).
Supplementary files format and content: feature_counts_output_KIC5.xlsx is the output of Feature counts and contains ID (GeneID), chromosome (Chr), and the counts (number of reads), for each mapped gene from each RNAseq sample.
|
| |
|
| Submission date |
May 27, 2022 |
| Last update date |
Nov 29, 2022 |
| Contact name |
Caroline Simmons |
| Organization name |
University of South Florida
|
| Department |
Center for Global Health and Infectious Diseases Research
|
| Lab |
John Adams Laboratory
|
| Street address |
3720 East Spectrum Blvd St. 404
|
| City |
Tampa |
| State/province |
FL |
| ZIP/Postal code |
33620 |
| Country |
USA |
| |
|
| Platform ID |
GPL23997 |
| Series (1) |
| GSE205012 |
Protein KIC5 is a novel regulator of artemisinin stress response in the malaria parasite Plasmodium falciparum |
|
| Relations |
| BioSample |
SAMN28700475 |
| SRA |
SRX15484741 |
