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| Status |
Public on Sep 16, 2023 |
| Title |
AP2-XII-1-KD-HA_HA-antibody_UT |
| Sample type |
SRA |
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| Source name |
intracellular parasites
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| Organism |
Toxoplasma gondii |
| Characteristics |
strain: RHKU80_AP2-XII-1-KD-HA
chip antibody: HA antibody (Cell Signaling)
dev. stage: tachyzoites
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| Treatment protocol |
HFF cells were grown to confluence and infected with RHKU80 single AP2XII-1, AP2XI-2 mAiD or the double AP2-XII-1/AP2XI-2 mAiD strains. Genome-wide Chip sequencing was performed to detect AP2-XII-1 and/or AP2-XI-2, HDAC3, MORC in two conditions; left untreated (UT) and treated with IAA (indole-3-acetic acid) for 24 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 RHKU80 strains 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 receiving demultiplexed fastq files from the sequencing service provider (ArrayStar), reads were aligned to Toxoplasma gondii reference genome (TGME49) using BOWTIE2. Aligned reads were used for peak calling of the ChIP regions using MACS2. Statistically significant ChIP-enriched regions (peaks) were identified individually using a p-value threshold of 10-4. BigWig datasets were generated using bedGraphToBigWig to convert the bedgraph file generated by MACS2
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).
Assembly: 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 individually, using a p-value threshold of 10-4. Te BigWig format file containing the ChIP-enriched regions and 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 |
Jan 13, 2023 |
| Last update date |
Sep 16, 2023 |
| Contact name |
Christopher Swale |
| E-mail(s) |
[email protected]
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| Organization name |
INSERM
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| Department |
U1209 - Institute of Advanced Biosciences
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| Lab |
Hakimi Lab
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| Street address |
Batiment Jean Roger
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| City |
La Tronche |
| ZIP/Postal code |
38700 |
| Country |
France |
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| Platform ID |
GPL23466 |
| Series (2) |
| GSE222819 |
In vitro production of cat-restricted Toxoplasma pre-sexual stages by epigenetic reprogramming [ChIP-seq] |
| GSE222835 |
In vitro production of cat-restricted Toxoplasma pre-sexual stages by epigenetic reprogramming |
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| Relations |
| BioSample |
SAMN32727478 |
| SRA |
SRX19026616 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6932717_Chip-RH_AP2-XII-1-KD-HA_HA-antibody_UT_MACS2.bw |
81.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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