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| Status |
Public on Aug 03, 2021 |
| Title |
PfAP2-G_GFP_WT_T_rep1_INPUT_ChIP-seq |
| Sample type |
SRA |
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| Source name |
PfAP2-G_GFP_WT_T_rep1_INPUT_ChIP-seq
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| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Trophozoite (30-40hpi)
strain: Pf 3D7
chip antibody: none
genotype: wild type
host: Homo sapiens
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| Treatment protocol |
PfAP2-G5-GFP::WT, PfAP2-G-GFP::WT, PfAP2-G-GFP::pfap2-g5_trun parasite lines were synchronized twice in two continuous intraerythrocytic cycles by 5% sorbitol solution to harvest trophozoite stage parasites. PfAP2-G5-GFP::WT line was synchronized twice with 5% sorbitol solution followed by a percoll treatment in the cycle before the cultures were set up for gametocytogenesis assay. Then the highly synchronized ring-stage parasites were set up at 2% parasitemia (4% haematocrit). After re-invasion, 50 mM N-acetyl-D-glucosamine was added into the cultures to kill the asexual replicating forms after sexual commitment to harvest G0-G1 stage parasites.
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| 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Synchronized PfAP2-G5-GFP::WT, PfAP2-G-GFP::WT, PfAP2-G-GFP::pfap2-g5_trun parasites at different stages were harvested and cross-linked immediately with 1% paraformaldehyde (Sigma) by rotating for 10 min at 37 ℃, then quenched with 0.125 M glycine for 5 min. The culture was resuspended with 1 x PBS and parasites were released from iRBC with 0.15% saponin for 10- 15 min on ice. The washed nuclei were isolated by incubation with 2 ml of Lysis Buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA pH8.0, 0.1mM EGTA pH8.0, 1mM DTT, 0.25% NP40, 1 x protease inhibitors cocktail) for 30 min on ice, and followed by dounce homogenization for 100 strokes. Afterwards the nuclei were resuspended in 200 μl of SDS Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH8.0) for sonication to generate 200–500 bp fragments in length. The sonicated chromatin was diluted ten-fold with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.0, 150 mM NaCl, 1 x protease inhibitors cocktail) and precleared with Protein A/G magnetic beads (Pierce) for two hours. The precleared chromatin supernatant was incubated with 1 μg rabbit anti-GFP (Abcam, ab290) or rabbit IgG (Abcam) as control and Protein A/G magnetic beads at 4 ℃ overnight with a small volume of non-immunoprecipitated input material kept separately. After extensive washes, protein-DNA complexes were eluted with Elution Buffer (1% SDS, 0.1 M NaHCO3). Crosslinking was reversed by incubating overnight at 45°C and DNA was treated with RNase A at 37°C for 30 min and Proteinase K at 45°C for two hours. ChIP-DNA was extracted using MinElute PCR purification kit (Qiagen, 28006).
ChIP-DNA was end-repaired (Epicentre No.ER81050), adding protruding 3’ A base (NEB No.M0212L), adapter ligation(NEB No.M2200L), size selection and amplification of libraries(KAPA Biosystems, KB2500) with a PCR program: 1 min at 98 ℃, 12 cycles of 10 s at 98 ℃, 1 min at 65 ℃; finally extended 5 min at 65 ℃.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Low-quality and the 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 Bowtie2 (v2.3.4.3) with parameters: -N 0 --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.
Next, the duplicated reads were removed by markdup from sambamba (v0.6.8) .
MACS2 (v2.1.1) was used to perform peak calling with the p-value cutoff of 0.01.
Genome_build: Pf 3D7 v32
Supplementary_files_format_and_content: 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.
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| Submission date |
May 03, 2020 |
| Last update date |
Aug 03, 2021 |
| 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) |
| GSE149773 |
A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [ChIP-Seq TF] |
| GSE149774 |
A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) |
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| Relations |
| BioSample |
SAMN14823826 |
| SRA |
SRX8243391 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4512254_PfAP2-G_GFP_WT_T_rep1_INPUT_ChIP-seq.bw |
15.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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