|
| Status |
Public on Nov 15, 2023 |
| Title |
Trophozoites, RNAseq_MorcKD_Mel1 |
| Sample type |
SRA |
| |
|
| Source name |
whole organism
|
| Organism |
Plasmodium falciparum |
| Characteristics |
isolate: 3D7
Stage: trophozoites
genotype: PfMORC-KD
treatment: 100nM Melatonin
time: 5 hours
tissue: whole organism
|
| Treatment protocol |
To induce PfMORC knockdown, tightly synchronized parasites at young trophozoite stage (22hpi) were treated with 2.5 mM glucosamine (GlcN) till next cycle. Parasites at 34hpi were harvested for total RNA extraction and stored in Trizol reagent. For melatonin assay, when majority of parasites reached to 28hpi, 100 nM melatonin was added in culture for 5h and then parasites were harvested and stored in Trizol reagent.
|
| Growth protocol |
The P. falciparum strains were cultured at 37°C in RPMI 1640 medium supplemented with 0.5% Albumax II (Gibco). Parasites were grown under a 5% CO2, 5% O2, and 90% N2 atmosphere. Cultures were synchronized with 5% sorbitol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted following the manufacturer’s protocol, and an RNA cleanup kit (Qiagen) was used to achieve high-purity RNA. The isolated RNA was quantified using a NanoDrop ND-1000 UV/Vis spectrophotometer, and RNA quality was determined using an RNA ScreenTape System (Agilent 2200 TapeStation). Total RNA (10000 ng) from each sample was stabilized in RNAStable (Biomatrica) and sent to the Micromon Genomics facility at Monash University for next-generation sequencing.
RNA samples were prepared using the MGITech RNA Directional Library Prep Kit V2 (Item No. 1000006385), as per the manufacturer's instructions, with the following parameters: input RNA: 50 ng, fragmentation: target of 200 bp-400 bp, 87 °C for 6 mins, adapter clean-up: 200 bp-400 bp and library amplification cycles: 16. Libraries were assessed for quantity using the Invitrogen Qubit and dsDNA HS chemistry (Item No. Q33230) and quality/quality using the Agilent Fragment Analyser 5200 with the HS NGS Fragment Kit (Item No. DNF-473-0500). The libraries were pooled in equimolar concentrations and sequenced using an MGITech DNBSEQ-G400RS sequencing instrument with High-Throughput Sequencing Set (FCL PE100, Item No. 1000016950) chemistry.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
DNBSEQ-G400 |
| |
|
| Description |
MS-25-Mel-1
RNAseq of trophozoites stage Plasmodium falciparum PfMORC transgenic knockdown parasite treated with 100nM melatonin for 5 hours
|
| Data processing |
The quality of the RNA-seq libraries was evaluated using the FastQC tool. Next, we used Salmon (V1.9.0) quant with default arguments to quantify the expression of all transcripts in the PlasmoDB release-58 Pfalciparum3D7 genome. The transcript expression was summarized to gene-level expression with tximport 1.22.0. Finally, the gene counts were used to detect differentially expressed genes (DEGs) with DESeq2 (1.34.0). Furthermore, only genes with an >1 log2 fold change and adjusted p value <0.1 were considered significant for further analysis.
Assembly: PlasmoDB release-58 Pfalciparum3D7
Supplementary files format and content: PfMORC_gene_TPM.tsv, sf files
|
| |
|
| Submission date |
Aug 21, 2023 |
| Last update date |
Nov 15, 2023 |
| Contact name |
Maneesh Kumar Singh |
| E-mail(s) |
manish.sings@gmail.com
|
| Organization name |
University of Sao Paulo
|
| Street address |
Av. Prof. Lineu Prestes, 580
|
| City |
Sao Paulo |
| State/province |
SP |
| ZIP/Postal code |
05508-000 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL33701 |
| Series (1) |
| GSE241313 |
Plasmodium falciparum MORC protein modulates gene expression through interaction with heterochromatin |
|
| Relations |
| BioSample |
SAMN37096302 |
| SRA |
SRX21479844 |
