|
| Status |
Public on Jan 01, 2020 |
| Title |
PfRrp6-DD-1C-GlcN+_T_rep2_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
PfRrp6-DD-1C-GlcN+_T_rep2_RNA-seq
|
| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Trophozoite
strain: Pf 3D7
genotype: PfRrp6-DD-GlcN+
host: Homo sapiens
|
| Treatment protocol |
Parasite cultures were synchronized by repeated sorbitol and percoll-sorbitol treatments ((Lambros and Vanderberg, 1979)
|
| Growth protocol |
Parasite-infected red blood cells were cultured in T75 flask at 2% hematocrit in the presence of 5% O2 and 5% CO2 (rest N2) in RPMI 1640 supplemented with 25mM Hepes, 50mg/l Hypoxanthine, 0.21% NaHCO3, 0.5% Albumax I and 20mg/l Gentamicin. The vector line was selected and cultured with 2.5nM WR99210. The 5mM GlcN was added in the inducible knock down culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Highly synchronous parasites were harvested at the ring stage (10 h after invasion), Total RNA was isolated using TRIzol (Life) according to the manufacturer’s manual and further purified using the Direct-zol RNA Kit (ZYMO RESEARCH) for removal of gDNA. Residual gDNA was digested with TURBO DNA-freeTM DNaseI (Ambion) and the RNA was quantified by NanoDrop.
The RNA isolation, mRNA enrichment and library construction were performed as described using 15 cycles of library amplification. The mRNA was enriched 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 in accordance with an KAPA Stranded mRNA-Seq Kit Illumina platform (KAPABIOSYSTEMS KK8421-96 libraries).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
PfRrp6-DD clone line PfRrp6-DD-1C cultured with 5 nM GlcN
RRP6-DD-GlcN+_rep1_rep2_Raw_Counts_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 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.
|
| |
|
| Submission date |
Aug 15, 2019 |
| Last update date |
Jan 01, 2020 |
| 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) |
| GSE133236 |
RNA_seq data for Rrp6 project analysis in malaria (Plasmodium falciparum) |
| GSE133241 |
Rrp6-mediated heterochromatin surveillance secures antigenic variation and sexual commitment of human malaria parasites |
|
| Relations |
| BioSample |
SAMN12585578 |
| SRA |
SRX6721824 |
