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| Status |
Public on May 17, 2024 |
| Title |
H3K4me2, PI, REP2 |
| Sample type |
SRA |
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| Source name |
PI
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A549 (ATCC CCL-185)
cell type: lung carcinoma epithelial cells
chip antibody: ChIP-grade Abcam ab32356
infection: PI
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| Treatment protocol |
Cells were cross-linked with 1% formaldehyde 8 min at room temperature, followed by quenching with 0,125 M glycine for 5 min. After two washes in PBS, cells were collected by scraping then pelleted and lysed on ice for 5 min in 0.25% Triton X-100, 10 mM Tris-HCl (pH 8), 10 mM EDTA, 0.5 mM EGTA and proteases inhibitors. The soluble fraction was eliminated by centrifugation and chromatin was extracted with 250 mM NaCl, 50 mM Tris-HCl (pH 8), 1 mM EDTA, 0.5 mM EGTA and proteases inhibitors cocktail for 30 min on ice. Chromatin was resuspended in 1% SDS, 10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.5 mM EGTA and proteases inhibitors cocktail, then fragmented by sonication (10 cycles of 30 sec ‘on’ and 30 sec ‘off’) using Bioruptor Pico (Diagenode). Sheared chromatin was cleared by centrifugation, sampled for the shearing efficiency analysis with High Sensitivity DNA kit (Agilent, 5067-4626) using 2100 Bioanalyzer instrument (Agilent). 2 μg of antibodies ChIP-grade (H3K4me2, H3K4me3, H3, Normal Rabbit IgG). 2% of ChIP sample volume was reserved to serve as input. Diluted chromatin was then added to antibody bound DiaMag beads and incubated at 4˚C overnight with gentle rotation. ChIP samples were washed sequentially 5 min with buffer 1 (1% Triton X-100, 0.1% NaDOC, 150 mM NaCl, 10 mM Tris-HCl (pH 8)), buffer 2 (0.5% NP-40, 0.5% Triton X-100, 0.5 NaDOC, 150 mM NaCl, 10 mM Tris-HCl (pH 8)), buffer 3 (0.7% Triton X-100, 0.1% NaDOC, 250 mM NaCl, 10 mM Tris-HCl (pH 8)), buffer 4 (0.5% NP-40, 0.5% NaDOC, 250 mM LiCl, 20 mM Tris-HCl (pH 8), 1 mM EDTA), buffer 5 (0,1% NP-40, 150 mM NaCl, 20 mM Tris-HCl (pH 8), 1 mM EDTA) and buffer 6 (10 mM Tris-HCl (pH 8), 1 mM EDTA).
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| Growth protocol |
The human alveolar epithelial cell line A549 (ATCC CCL-185) were cultured in F-12K culture medium (Gibco™ 21127030) supplemented with 10% fetal calf serum (FCS) and 1% L-glutamine and incubated at 37 °C in a humidified atmosphere with 5% CO2. Bacteria (pneumococcus) were diluted in serum-low cell culture medium (0,25% FCS) to get a multiplicity of infection of 20:1 (MOI 20). After 3h of infection (primary), cells were washed twice with PBS to remove the unattached bacteria, and then either collected for analysis, or cultured in medium with penicillin-streptomycin (10 microg /ml) and gentamicin (200 microg /ml) for later time point (PI).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP and input samples were treated with IPure Kit (Diagenode, C03010015) followed protocol according to manufacturers’ instructions. ChIP and input DNA were quantified with Qubit 4 fluorometer (ThermoFisher). The libraries were performed with 10 ng each sample.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Complete analysis from the raw sequencing data to the differential marking analysis was done following the ePeak approach (cite https://doi.org/10.1093/nargab/lqac041).
Default parameters were used for the filtering, mapping of reads and narrow peak calling.
For H3K4me2, reproducible peaks were determined using the intersection approach, requiring at least 40% length overlap between replicates. For H3K4me3, reproducible peaks were determined using the IDR method. Coverage per replicate and for pooled replicates was calculated using nibs of 10bp and the CPM normalization method.
Assembly: hg19
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)
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| Submission date |
Apr 20, 2023 |
| Last update date |
May 17, 2024 |
| Contact name |
Claudia Chica |
| Organization name |
Institut Pasteur
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| Lab |
Biostatistics and Bioinformatics Hub
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| Street address |
25-28 Rue du Dr Roux
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| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE230141 |
Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3 [ChIPseq] |
| GSE230142 |
Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3 |
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| Relations |
| BioSample |
SAMN34264769 |
| SRA |
SRX20022143 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7188132_H3K4me2_PI_REP2_hg19.bw |
232.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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