|
| Status |
Public on Apr 05, 2024 |
| Title |
DoziKo_R1_20h |
| Sample type |
SRA |
| |
|
| Source name |
Plasmodium parasite
|
| Organism |
Plasmodium falciparum |
| Characteristics |
genotype: deltapfdozi
Stage: early-trophozoite
treatment: no treatment
|
| Treatment protocol |
To generate stressed schizonts, ring-stage PfDOZI::GFP parasites were synchronized twice in two successive IDCs by 5% D-sorbitol treatment. The synchronized trophozoites were then used to set up culture at 2.0–2.5% parasitemia and 3% hematocrit. On the following day, when the parasitemia reached 10–12%, two-third of the medium was replaced by the fresh medium to generate stressed schizonts.
|
| Growth protocol |
3D7 strains of P. falciparum were cultured in type O+ human RBCs at 5% hematocrit in RPMI 1640 medium supplemented with 25 mM HEPES, 50mg/L hypoxanthine, 25 mM NaHCO3, 0.5% Albumax II and 40 mg/ml gentamicin sulfate. The culture was maintained at 37°C in a gas mixture of 5% CO2, 3% O2 and 92% N2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Parasites of designated developmental stages were harvested after lysis of the RBC membrane with 0.1% (v/v) saponin in PBS. Total RNA was isolated from the purified parasites using Quick-RNA MiniPrep kit (Zymo Research). The RNA was treated with the ezDNaseTM Enzyme (Invitrogen) to remove contaminating genomic DNA. RNA pellets were dissolved in 50 µL RNase-free water, and the RNA integrity was checked using the Agilent Bioanalyzer.
RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. RNA was fragmented by heating at 94 °C for 8 min, and cDNA was synthesized using reverse transcriptase with random primers. Afterward, 3’ dTMP adapters were ligated to the 3’ dAMP library fragments. The quality of the barcoded libraries was validated using the Agilent Bioanalyzer. The DNA 1000 Reagents were used to quantify the library sizes and confirm the absence of primer dimers. Libraries were quantified using a KAPA Universal Library Quantification Kit (Roche), and library concentrations were adjusted for library size. Libraries were sequenced on an Illumina HiSeq 2500 in the Rapid Run mode using 100 nt single read sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
pfdozi_KO_20h.xlsx
|
| Data processing |
Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained.
Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2
Differentially expressed genes are determined by DESeq2 with Padj < 0.01 and absolute log2 fold change higher than 1.
The transcriptional profiles are normalized by TPM for multi-time points or condition comparisons.
Genome_build: pf3D7_V3.0.fasta
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
|
| |
|
| Submission date |
Nov 17, 2021 |
| Last update date |
Apr 05, 2024 |
| Contact name |
JUN MIAO |
| E-mail(s) |
jmiao1@usf.edu
|
| Phone |
8139747374
|
| Organization name |
University of South Florida
|
| Department |
Internal Medicine
|
| Lab |
Jun Miao and Liwang Cui
|
| Street address |
3720 Spectrum Boulevard, MDC84
|
| City |
Tampa |
| State/province |
Florida |
| ZIP/Postal code |
33612 |
| Country |
USA |
| |
|
| Platform ID |
GPL21078 |
| Series (1) |
| GSE189034 |
Global transcriptional changes of Δpfdozi parasite in different stages and conditions |
|
| Relations |
| BioSample |
SAMN23243020 |
| SRA |
SRX13158239 |
