|
| Status |
Public on Aug 07, 2023 |
| Title |
△AP2-MRPKO_40hpi_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Plasmodium falciparum II3 strain
|
| Organism |
Plasmodium falciparum |
| Characteristics |
genotype: deltaAP2-MRPKO
strain: II3
time point: 40hpi
|
| Treatment protocol |
DiCre recombinase-mediated excision was induced by adding rapamycin to the culture of ring-stage parasites at a final concentration of 10 nM.
|
| Growth protocol |
Parasites were maintained in human A+ erythrocytes at 37 °C in RPMI 1640 medium containing AlbumaxII (Invitrogen) supplemented with 2 mM L-glutamine. Parasites were either synchronized by sorbitol treatment or by purifying mature schizont stages using 70% (v/v) isotonic Percoll (GE Healthcare Life Sciences) before allowing reinvasion to occur, followed by sorbitol treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Parasite cultures (~0.5-2 ml depending on the asexual developmental stage) were pelleted at 2400 rpm for 3 min, lysed by adding 1ml of Trizol (Sigma), then immediately stored at -80°C until further use. Total RNA was isolated from Trizol lysed parasites according to manufacturer’s instructions (Life Technologies).
Strand-specific mRNA libraries were prepared from total RNA using TruSeq Stranded mRNA Sample Prep Kit LS (Illumina) according to the manufacturer’s instructions. Briefly, for each sample 100-300 ng of total RNA was used to prepare the libraries. PolyA+ mRNA was captured from total RNA using oligo-T attached to magnetic beads. First strand synthesis was performed using random primers followed by second strand synthesis where dUTP was incorporated in place of dTTP to achieve strand-specificity. Double stranded cDNA ends were ligated with adaptors and the libraries were amplified by PCR for 15 cycles, before sequencing the libraries on Illumina HiSeq-4000 platform with paired-end 150 bp read chemistry according to manufacturer’s instructions (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
bcl2fastq2 Conversion Software
The quality of the raw reads was assessed using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Low-quality reads and Illumina adaptor sequences from the read ends were removed using TrimmomaticR (Bolger et al., 2014). Processed reads were mapped to the P. falciparum 3D7 reference genome (release 40 in PlasmoDB- http://www.plasmoddb.org) using Hisat2 (Kim et al., 2015) (V 2.1.0) with parameter “—rna-strandness FR”. Counts per feature were estimated using FeatureCounts (Liao et al., 2014). Raw read counts data were converted to counts per million (cpm) and genes were excluded if they failed to achieve a cpm value of 1 in at least one of the three replicates performed. Library sizes were scale-normalized by the TMM method using EdgeR software (McCarthy et al., 2012) and further subjected to linear model analysis using the voom function in the limma package (Ritchie et al., 2015). Differential expression analysis was performed using DeSeq2 (Love et al., 2014). Genes with a fold-change greater than two and a false discovery rate corrected p-value (Benjamini-Hochberg procedure) < 0.05 were considered to be differentially expressed.
Genome_build: Plasmodium falciparum
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
|
| |
|
| Submission date |
Dec 07, 2021 |
| Last update date |
Aug 07, 2023 |
| Contact name |
AMIT KUMAR SUBUDHI |
| E-mail(s) |
amit.subudhi@kaust.edu.sa
|
| Phone |
+96540375986
|
| Organization name |
King Abdullah University of Science and Technology
|
| Department |
BESE
|
| Lab |
Pathogen Genomics
|
| Street address |
Level4, Builiding, 2, KAUST
|
| City |
Thuwal |
| State/province |
Thuwal |
| ZIP/Postal code |
239556900 |
| Country |
Saudi Arabia |
| |
|
| Platform ID |
GPL27825 |
| Series (2) |
| GSE190342 |
PfAP2-P DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages (RNA-seq) |
| GSE190519 |
PfAP2-P DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages |
|
| Relations |
| BioSample |
SAMN23721417 |
| SRA |
SRX13339423 |
