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| Status |
Public on Feb 10, 2017 |
| Title |
Pf_DCJ_H33Ty1_T30_BB2_xLCHIP |
| Sample type |
SRA |
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| Source name |
red blood cell stage, trophozoites (30hpi)
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| Organism |
Plasmodium falciparum |
| Characteristics |
strain: NF54 - DCJ clone
genomic modification: endogenous PfH3.3 (PF3D7_0617900) C-terminally tagged with Ty1 by single cross-over homologous recombination
development stage: trophozoites
chip antibody: monoclonal anti-Ty1 antibody (BB2)
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| Treatment protocol |
Prior to collection parasites were selected for var2CSA expression by affinity purification as in (Noviyanti et al., 2001). For the collection parasites were synchronized with sorbitol treatments and Percoll gradient centrifugations as described previously (Bártfai et al., 2010). During collections, medium was changed every 10 hours, but not less than 10 hours before collection to ensure optimal parasite growth. After 20 hours post invasion (hpi), medium volume was doubled. During each medium change parasite cultures were mixed to minimize growth differences within single culture flasks. Early ring, late ring, trophozoite and schizont stages were collected (10, 20, 30 or 40 hpi respectively).
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| Growth protocol |
Parasites were cultured in standard RPMI medium supplemented with 10% human serum (AB), 0.2% NaHCO3 and 2.5% human O+ red blood cells. Culturing occurred in 250 ml tissue culture flasks placed in candle jars or in an incubator with a gas composition of 3% O2, 4% CO2 and 93% N2 and incubated at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
xl anti-Ty ChIP (H3.3) protocol to extract and prepare the material: Parasite cultures used for cross-linked ChIP were immediately treated with 1% formaldehyde (final concentration) and incubated at 37°C for 10 min while shaking. Crosslinking reaction was quenched by adding 0.125M glycine (final concentration). After erythrocyte and parasite lysis using 0.05% saponin and a hypotonic buffer (10 mM Tris pH 8.0, 3 mM MgCl2 , 0.2% NP-40, Roche Protease Inhibitor Cocktail) respectively, formaldehyde-cross-linked nuclei were separated from cell debris using a 0.25 M sucrose buffer cushion as described in (Bártfai et al., 2010). Subsequent, crosslinked chromatin was prepared as described in (Kensche et al., 2015) with a few adjustments. In short, for generation of genome-wide PfH3.3 profiles, enzymatic MNase digestion of cross-linked chromatin was performed using digestion buffer (50mM Tris pH7.4, 4mM MgCl2, 1mM CaCl2, 0.075% NP40, Roche Protease Inhibitor Cocktail) with 0.5 U MNase (Worthington Biochemicals Corporation) in 150μl aliquots at 37 ºC. Optimal digestion times were empirically determined for each stage by test-digestion of a nuclear aliquot for varying times, to obtain primarily mono-nucleosomal DNA fragments. Digestion reactions were stopped by adding 150μl quenching solution (2% Triton X100, 0.6% SDS, 300 mM NaCl, 6 mM EDTA, Roche Protease Inhibitor Cocktail) and placing the samples on ice. Subsequently, digested chromatin was sonicated for 6 x 10 sec (setting low, BioruptorTM Next Gen, Diagenode) to free cross-linked chromatin from nuclear membranes. Approximately 200 ng of digested DNA-containing cross-linked chromatin was incubated in ChIP buffer (150mM NaCl, 20mM Tris pH 8.0, 2mM EDTA, 1% Triton X-100, 0.1% SDS, Roche Protease Inhibitor Cocktail ) with the respective antibody overnight at 4°C while rotating, followed by the addition of 10µl ProtA/G beads (Life Technologies, 10008D and 10009D) and further incubation at 4°C for 2 h. For PfH3.3 (anti-Ty1) four ChIP reactions were performed in parallel per life cycle stage to ensure sufficient amount of DNA for subsequent ChIP-seq (2 µg antibody /ChIP). After washing with three different buffers (buffer1, 1x: 20mM Tris pH 8.0, 2mM EDTA, 1% Triton-X100, 0.1% SDS, 150mM NaCl; buffer 2, 2x: 20mM Tris pH 8.0, 2mM EDTA, 1% Triton-X100, 0.1% SDS, 500mM NaCl and buffer 3, 2x: 10mM Tris pH 8.0, 1mM EDTA) immunoprecipitated chromatin was eluted using 1% SDS and 0.1M NaHCO3. Immunoprecipitated chromatin was de-cross-linked (1% SDS, 0.1M NaHCO3, 1M NaCl) overnight at 45 °C shaking, followed by DNA isolation via QIAquick column purification (Qiagen).
Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification
For each sequencing library generated from endogenously PfH3.3-Ty1 expressing DCJ P. falciparum parasites 3 ng of ChIP DNA were end repaired, extended with 3’ A-overhangs, and ligated to barcoded NextFlex adapters (Bio Scientific) (Hoeijmakers et al., 2011). Subsequent library amplification was performed under similar conditions as described previously (Kensche et al., 2015) with a few adjustments: First, 2x KAPA HiFi HotStart ready-mix (KAPA Biosystems) and NextFlex primers were used for 4 cycles of PCR amplification (98°C for 2min; 4 cycles of 98°C for 20sec, 62°C for 3min; 62°C for 5 min). Subsequently, amplified libraries were size selected for 270 bp (mono-nucleosomes + ~125bp NextFlex adapter) using 2% E-Gel Size Select Agarose Gels (Thermo Fisher Scientific) and again amplified as described above for 9 cycles. To deplete adapter dimers and clean-up the DNA, libraries were subsequently purified with Agencourt AMPure XP bead (Beckman Coulter) (1:1 library beads ratio) before sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava 1.8.2 software for basecalling.
Data were mapped with BWA samse (Version: 0.7.10-r789) to the P. falciparum 3D7 reference genome from PlasmoDB version 6.1 (http://www.plasmodb.org).
Sam files were converted to bam files, q15 filtered and filtered for unique reads
Normalized bedGraph files were generated using bedtools genomecov with the option –bga (regions with zero coverage are also reported) and normalized using the option -scale and the scaling factor ‘1000000/amount of unique reads’. These normalized files were used to generate log2 PfH3.3-ChIP-over-H3core-ChIP ratio tracks.
Genome_build: PlasmoDB Plasmodium falciparum 3D7 release 6.1
Supplementary_files_format_and_content: ratio bedGraph.gz files show the coverage as log2-ratio of PfH3.3-ChIP-over-PfH3core-ChIP calculated using normalized bedGraph files (T*_H33_H3core.Bedgraph.gz) (unique reads only, normalized to tag counts per million)
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| Submission date |
Apr 20, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sabine Fraschka |
| Organization name |
Radboud University Nijmegen
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| Department |
Molecular Biology
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| Lab |
Richárd Bártfai
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| Street address |
Geert Grooteplein 28
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL16607 |
| Series (1) |
| GSE80466 |
Histone variant H3.3 demarcates GC-rich coding and subtelomeric sequences and serves as a potential memory mark for virulence gene expression in P. falciparum |
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| Relations |
| BioSample |
SAMN04874683 |
| SRA |
SRX1715898 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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