|
| Status |
Public on Aug 03, 2021 |
| Title |
WT_NF54_R_rep2_RNA-seq (control for pfap2-g5_trun) |
| Sample type |
SRA |
| |
|
| Source name |
WT_NF54_R_rep2_RNA-seq (control for pfap2-g5_trun)
|
| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Ring
strain: Pf NF54
genotype: wild type
host: Homo sapiens
|
| Treatment protocol |
Parasite lines were synchronized twice in two continuous intraerythrocytic cycles by 5% sorbitol solution followed by a percoll treatment in the cycle to obtain asexual ring (10-15 hpi), trophozoite (25-30 hpi), schizont (40-45 hpi) at continuous timepoints. At the same time, the highly synchronized ring-stage parasites were set up at 2% parasitemia (4% haematocrit) to perform gametocytogenesis assay . After re-invasion, 50 mM N-acetyl-D-glucosamine was added into the cultures to kill the asexual replicating forms after sexual commitment. The growth of the parasites was monitored by making Giemsa smears every day until mature gametocytes were observed in the culture by Giemsa staining of thin blood smears. Parasites were harvested at day 2, 4, 6, 8, 10 during the process of gametocyte induction.
|
| Growth protocol |
Parasites were cultured in medium containing 0.5% Albumax II with O type fresh red blood cells, and a gas phase with 5% O2, 5 % CO2 at 37 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from parasites with a combination of TRIzol (Invitrogen) treatment and using a Direct-zol RNA Kit (Zymo Research) as manufacturer’s instructions.
Library preparation for strand-specific RNA-seq was carried out by poly(A) selection with the KAPA mRNA Capture Beads (KAPA), and fragmented to about 300–400 nucleotides (nt) in length, then all subsequent steps were performed according to a KAPA Stranded mRNA-Seq Kit Illumina platform (KK8421).
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
WT_vs_pfap2-g5_trun_NF54_rep2_FPKM_sense_and_antisense_RNA.xlsx
|
| Data processing |
Low-quality and adaptor sequences were trimmed from the reads using cutadapt (v1.16) with parameters: -a AGATCGGAAGAGC -A AGATCGGAAGAGC --trim-n -m 75 -q 20,20.
Then, the reads were mapped to the Plasmodium falciparum 3D7 genome (Pf 3D7 v32, obtained from PlasmoDB) using Hisat2 strand-specific mode (v2.1.0) with parameters: --rna-strandness RF --dta --no-discordant --no-mixed --no-unal.
The Samtools (v1.9) was used to filter low mapping quality reads and transfer the mapping results from sam format to position sorted bam format.
Mapped reads were subsequently assembled into transcripts guided by the PlasmoDB gff annotation files (Pf 3D7 v32) using featureCounts (v1.6.1) with parameters: -M -p -B -C for all; -s 2 for sense transcripts; -s 1 for antisense transcripts.
The bam files were converted to bigwig files using bamCoverage from the deeptools suite (v3.1.3) with parameters: --normalizeUsing RPKM --binSize 10 --smoothLength 30 -ignore Pf3D7_API_v3 Pf_M76611
Genome_build: Pf 3D7 v32
Supplementary_files_format_and_content: The raw counts were calculated by featureCounts as described above. Then FPKM of each gene was calculated in R.
|
| |
|
| Submission date |
May 03, 2020 |
| Last update date |
Aug 03, 2021 |
| Contact name |
Shijun Shen |
| E-mail(s) |
xiaoshen19930901@163.com
|
| Phone |
+86-21-13795384821
|
| Organization name |
Tongji University
|
| Department |
Bioninformatics
|
| Lab |
Jiang Lab
|
| Street address |
1239 Siping Road, Shanghai, P.R. China
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
| |
|
| Platform ID |
GPL26835 |
| Series (2) |
| GSE149772 |
A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [RNA-Seq] |
| GSE149774 |
A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) |
|
| Relations |
| BioSample |
SAMN14823719 |
| SRA |
SRX8243306 |
