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| Status |
Public on Jan 01, 2020 |
| Title |
CP-PfRrp6-DD-1C_R_rep1_ChIRP-seq |
| Sample type |
SRA |
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| Source name |
Complementary-Probe-PfRrp6-DD-1C_R_rep1_ChIRP-seq
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| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Ring
strain: Pf 3D7
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 (15 hr post invasion ±5 hr) synchronized 3D7-G7 and the PfRrp6-DD-1C line were used in ChIRP assay. Parasites culture (1x109 rings) were cross-linked as the ChIP arrays. After three washes with cold 1 x PBS, the parasite pellets were lysed with the swelling buffer (0.1M Tris pH 7.0, 10mM KOAc, 15mM MgOAc, 1% NP-40, 1mM DTT, protease inhibitor, and RNase inhibitor) at 4℃ for 10min with rotator. After centrifuge at 10,000 rpm for 10 min at 4 ℃, the parasites were resuspended in 150μl SDS Lysis Buffer ( 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0 ) 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. Chromatin is diluted in 2 times volume of hybridization buffer (500mM NaCl, 1%SDS, 100mM Tris 7.0, 10mM EDTA, 15% Formamide, add DTT, PMSF, P.I, and RNase inhibitor fresh). 100 pmol probes were added to each reaction, which was mixed by end to-end rotation at 37℃ for 4 hours. Streptavidin-magnetic T1 beads were washed three times in lysis buffer, blocked with 500ng/μl yeast total RNA and 1mg/ml BSA for 1 hour at 37℃, and washed three times again in lysis buffer before resuspended in its original volume. 100μl washed/blocked T1 beads were added per 100 pmol of probes, and the whole reaction was mixed for another 45min at 37℃. Beads : biotin -probes : RNA : chromatin products were captured by magnets (Invitrogen) and washed five times with 40x beads volume of wash buffer (2 x SSC, 0.5% SDS, add DTT and PMSF fresh). After last wash buffer was removed carefully with P-10 pipette, beads were resuspended in 3x original volume DNA elution buffer (50mM NaHCO3, 1%SDS, 200mM NaCl), and DNA was eluted with 100μg/ml RNase A and 0.1U/μl RNase H. RNase elution was carried out twice at 37 ℃ with end-to-end rotation. Then chromatin was reverse-crosslinked with 0.2U/μl protease K at 65℃ overnight. DNA was then extracted with equal volume of phenol : chloroform : isoamyl. Eluted DNA was subject to high-throughput sequencing.
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 |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
Complementary probes were designed on the sense strand of RUF6 ncRNAs
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| Data processing |
Library strategy: ChIRP-seq
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) .
ChIRP peaks were predicted by MACS2 (v2.1.1) with Q-value < 0.01 and the parameter: -c.
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) |
| GSE133239 |
ChIRP_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 |
SAMN12128797 |
| SRA |
SRX6357992 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3903882_CP-PfRrp6-DD-1C_R_rep1_ChIRP-seq.bw |
15.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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