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| Status |
Public on Jan 01, 2020 |
| Title |
PfRrp4_T_rep1_RIP-seq |
| Sample type |
SRA |
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| Source name |
transgenic parasite line of Pfrrp4-HAx3-Ty1x3,no-crosslink
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| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Trophozoite
strain: Pf 3D7
rip antibody: mouse anti-Ty1,Sigma SAB4800032-50 ug
genotype: wild type
host: Homo sapiens
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| Treatment protocol |
Parasite cultures were synchronized by repeated sorbitol and percoll-sorbitol treatments ((Lambros and Vanderberg, 1979)
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| 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 GFP parasite line was cultured in presence of 2.5nM WR99210 (WR).
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| Extracted molecule |
total RNA |
| Extraction protocol |
An equivalent of 5 x 109 PfRrp6, PfRrp4 and GFP parasites were grown to 15–20 h ring stages in RPMI complete medium. Free parasites were collected by saponin lysis. The resulting parasite pellet was lysed under non-denaturing conditions (50mM Tris-Cl pH7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100/NP-40, Proteinase inhibitor, RNase inhibitor) for 2h at 4℃ with rotator, then the supernatant was collected by centrifugation at 13,000rpm for 15min at 4℃, and subjected to immunoprecipitation analysis with mouse anti-Ty1 antibodies for 3h at 4 ℃, then incubated with the protein G magnetic beads (Thermo) overnight at 4 ℃. After washing the beads twice with IPP500 (500 mM NaCl, 10 mM Tris-Cl, pH 8.0, 0.05% NP-40, Proteinase inhibitor, RNase inhibitor) and once with 1 x PBS, bounded RNA was eluted by TRIzol reagent for 10min with rotator at 4℃, then RNA was extracted by phenol-chloroform method and treated with DNaseI for 10min at room temperature. IPed RNA was directly used to prepare strand-specific RNA-seq libraries(see below), without poly(A) enrichment. A minimum of two biological replicates was sequenced for each experimental and control immunoprecipitation.
The IPed RNA was 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).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| 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 50 -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.
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| Submission date |
Jun 24, 2019 |
| Last update date |
Jan 01, 2020 |
| Contact name |
Shijun Shen |
| E-mail(s) |
xiaoshen19930901@163.com
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| Phone |
+86-21-13795384821
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| Organization name |
Tongji University
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| Department |
Bioninformatics
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| Lab |
Jiang Lab
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| Street address |
1239 Siping Road, Shanghai, P.R. China
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| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
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| Platform ID |
GPL26835 |
| Series (2) |
| GSE133237 |
RIP_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 |
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| Relations |
| BioSample |
SAMN12128782 |
| SRA |
SRX6357960 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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