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| Status |
Public on Jan 01, 2020 |
| Title |
PfRrp6-DD-1C_HM-HP1-input_R_rep1_ChIP-seq |
| Sample type |
SRA |
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| Source name |
PfRrp6-DD-1C_HM-HP1-input_R_rep1_ChIP-seq
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| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Ring
strain: Pf 3D7
chip antibody: none
genotype: PfRrp6-DD
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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Ring-stage (10 hr post invasion ±5 hr) synchronized 3D7-G7 and PfRrp6-DD-1C lines were used in ChIP assay. Parasites culture (1x109 rings) was harvested and cross-linked immediately with 1% paraformaldehyde with rotation for 10 min at 37℃, and quenched with 0.125 M glycine for 5 min on ice. The fixed culture was washed twice with cold 1xPBS, and the parasites were released from iRBC with 0.15% saponin for 15min on ice. After washed with cold 1xPBS, nuclei were isolated by incubation with 2 ml cold 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, 1x protease inhibitors cocktail) for 30 min on ice, and followed by dounce homogenization for 200 strokes. After centrifuge at 10,000 rpm for 10 min at 4 ℃, the nuclei was resuspended in 150μl SDS Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.0) for sonication. Chromatin was sheared into 200-500 bp fragments with Bioruptor (BioruptorTM UCD-200) of highest power for 4x4 min at 30 sec intervals. The resulting chromatin was diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.0, 150 mM NaCl, 1x protease inhibitors cocktail). Pre-clear the chromatin solution with 50 μl/ml of salmon sperm DNA/protein A/G agarose slurry for 2h at 4℃ with agitation. After centrifugation, per 250μl chromatin supernatant was incubated with 3μg of Rabbit polyclonal H3K9me3 (abcam, ab8898), 3μg of Rabbit polyclonal H3K9Ac (Millipore, 07-352) and 3μg of Rabbit polyclonal HP1 for each IP overnight at 4℃ and rabbit IgG (Sigma) as control. To collect immune complexes, the sample was incubated with 50μl of Salmon sperm DNA/protein A/G slurry for 2h at 4℃ with agitation. After extensive washes, immunoprecipitated chromatin was eluted with ChIP Elution Buffer (1% SDS, 0.1 M NaHCO3), and reverse crosslinked at 65℃ for 16 h in the presence of 200 mM NaCl, then added RNase A at 37℃ for 30min and incubated with the proteinase K at 45℃ for 2h. DNA was purified by Phenol/chloroform/isoamylic alcohol extraction for ChIP-seq.
To prepare sequencing libraries, 10 ng of input DNA, 3-5ng IPed DNA were end repaired, extended with 3′ A-overhangs and ligated to barcoded NextFlex adapters (Bio Scientific). Libraries were amplified using an optimized KAPA protocol using KAPA HiFi HotStart ready mix (KAPA Biosystems) and the following PCR program: 98°C for 3 min followed by 14 cycles of 98°C for 20 sec, 65°C for 30 s; 68°C for 30s; finally extended 68°C for 5min. Amplified libraries were size-selected for 300-600 bp using 2% agarose gels. ChIP-seq libraries were sequenced on the HiSeq X Ten machine.
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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 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) .
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 |
Jun 24, 2019 |
| Last update date |
Jan 02, 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) |
| GSE133238 |
ChIP_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 |
SAMN12128766 |
| SRA |
SRX6357979 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3903869_PfRrp6-DD-1C_HM-HP1-input_R_rep1_ChIP-seq.bw |
16.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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