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| Status |
Public on Dec 21, 2019 |
| Title |
Sample 1 (5 - ChIP 6): H4K31me1_UT |
| Sample type |
SRA |
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| Source name |
intracellular parasites
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| Organism |
Toxoplasma gondii |
| Characteristics |
strain: Pruku80_MORC_mAID_HA_KD
chip antibody: H4K31me1 - Home made antibody
dev. stage: tachyzoites
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| Treatment protocol |
HFF cells were grown to confluence and infected with Pruku80_MORC_HA or Pruku80_MORC_mAID_HA_KD strains. Genome-wide MORC and HDAC3 occupancy profiles in Pruku80_MORC_mAID_HA_KD left untreated (UT) and treated with IAA (indole-3-acetic acid) for 30 hours. Harvested intracellular parasites were crosslinked with formaldehyde (final concentration 1%) for 8 mins at room temperature and the crosslinking was stopped by addition of glycine (final concentration 0.125M) for 5 min at room temperature.
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| Growth protocol |
Toxoplasma gondii type II strains Pruku80_MORC_HA and Pruku80_MORC_mAID_HA_KD were maintained by serial passage Human foreskin fibroblast (HFF) monolayer under tachyzoite conditions in DMEM (Life) supplemented with 10% (vol/vol) FBS (Life) and 25 mM Hepes buffer, pH 7.2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked chromatin was lysed in ice-cold lysis buffer (50mM HEPES KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5%NP-40, 0.125% triton X-100, protease inhibitor cocktail) and sheared in shearing buffer (1mM EDTA pH8.0, 0.5mM EGTA pH8.0, 10mM Tris pH8.0, protease inhibitor cocktail) by sonication using a Diagenode Biorupter. Samples were sonicated, for 16 cycles (30 seconds ON and 30 seconds OFF), to 200-500 base-pair average size. Immunoprecipitation was carried out using sheared chromatin, 5% BSA, protease inhibitor cocktail, 10% triton X-100, 10% deoxycholate, DiaMag Protein A-coated magnetic beads (Diagenode) and antibodies (H4K5acK8acK12acK16ac, H4K31me1, HDAC3, and HA). A rabbit IgG antiserum was used as a control mock. After overnight incubation at 4°C on rotating wheel, chromatin-antibody complexes were washed and eluted from beads by using iDeal ChIP-seq kit for Histones (Diagenode) according to the manufacturer’s protocol. Samples were decrosslinked by heating for 4 hours at 65°C. DNA was purified by using IPure kit (Diagenode) and quantified by using Qubit Assays (Thermo Fisher Scientific) according to the manufacturer's protocol. For ChIP-seq, purified DNA was used to prepare libraries and then sequenced by Arraystar (USA, http://www.arraystar.com/).
ChIP-Sequencing library preparation was performed according to Illumina’s protocol Preparing Samples for ChIP Sequencing of DNA. Library Preparation: 10 ng DNA of each sample was converted to phosphorylated blunt-ended with T4 DNA polymerase, Klenow polymerase and T4 polymerase (NEB); An ‘A’ base was added to the 3' end of the blunt phosphorylated DNA fragments using the polymerase activity of Klenow (exo minus) polymerase (NEB); Illumina's genomic adapters were ligated to the A tailed DNA fragments; PCR amplification was performed to enrich ligated fragments using Phusion High Fidelity PCR Master Mix with HF Buffer (Finnzymes Oy). The enriched product of ~200-700 bp was cut out from gel and purified. Sequencing: The library was denatured with 0.1M NaOH to generate single-stranded DNA molecules, and loaded onto channels of the flow cell at 8pM concentration, amplified in situ using TruSeq Rapid SR cluster kit (#GD-402-4001, Illumina). Sequencing was carried out by running 100 cycles on Illumina HiSeq 4000 according to the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
untreated sample
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| Data processing |
Data Analysis: After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8). After passing Solexa CHASTITY quality filter, the clean reads were aligned to Toxoplasma gondii reference genome (Tgo) using BOWTIE (V2.1.0). Aligned reads were used for peak calling of the ChIP regions using MACS V1.4.0. Statistically significant ChIP-enriched regions (peaks) were identified by comparison of two samples, using a p-value threshold of 10-5. Then the peaks in each sample were annotated by the overlapped gene using the newest Toxoplasma gondii database. The EXCEL/BED format file containing the ChIP-enriched regions was generated for each sample.
Data visualization: The mapped 100 bp reads represent enriched DNA fragments by ChIP experiment. Any region of interest in the raw ChIP-seq signal profile can be directly visualized in genome browser. We use 10-bp resolution intervals (10-bp bins) to partition the stacked reads region, and count the number of reads in each bin. All the 10 bp resolution ChIP-seq profiles of each sample are saved as UCSC wig format files, which can be visualized in Toxoplasma gondii Genome Browser (http://protists.ensembl.org/Toxoplasma_gondii/Info/ Index).
Genome_build: Toxoplasma gondii genome (ME49 strain) file "ToxoDB-13.0_TgondiiME49_Genome.fasta" to upload at ToxoDB website : http://toxodb.org/common/downloads/release-13.0/TgondiiME49/fasta/data/
Supplementary_files_format_and_content: Statistically significant ChIP-enriched regions (peaks) were identified by comparison of two samples, using a p-value threshold of 10-5. Then the peaks in each sample were annotated by the overlapped gene using the newest Toxoplasma gondii database. The EXCEL/BED format file containing the ChIP-enriched regions was generated for each sample. We use 10-bp resolution intervals (10-bp bins) to partition the stacked reads region, and count the number of reads in each bin. All the 10 bp resolution ChIP-seq profiles of each sample are saved as UCSC wig format files, which can be visualized in Toxoplasma gondii Genome Browser (http://protists.ensembl.org/Toxoplasma_gondii/Info/ Index).
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| Submission date |
Aug 20, 2019 |
| Last update date |
Dec 23, 2019 |
| Contact name |
Mohamed-ali HAKIMI |
| E-mail(s) |
[email protected]
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| Phone |
(33)476637469
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| Organization name |
CNRS
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| Department |
UMR5309
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| Lab |
IAB - HAKIMI Team
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| Street address |
Domaine de la Merci, Campus Santé
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| City |
LA TRONCHE |
| State/province |
Grenoble |
| ZIP/Postal code |
38700 |
| Country |
France |
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| Platform ID |
GPL23466 |
| Series (1) |
| GSE136060 |
A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment |
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| Relations |
| BioSample |
SAMN12608809 |
| SRA |
SRX6743985 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4040357_H4K31me1_UT.wig.gz |
16.9 Mb |
(ftp)(http) |
WIG |
| GSM4040357_H4K31me1_UT_alignment.bed.gz |
101.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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