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| Status |
Public on Jul 14, 2023 |
| Title |
Input2_D |
| Sample type |
SRA |
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| Source name |
mammary glands
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| Organism |
Mus musculus |
| Characteristics |
tissue: mammary glands
cell line: Established from mammary epithelial cells
cell type: epithelial cells
genotype: MIC/EZH2wt/wt
treatment: Treat with doxycycline for 8 days
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| Treatment protocol |
EZH2wt/wt MIC organoids were treated with doxycycline for 8 days prior to fixation and crosslinking with 1% formaldehyde for 10 min. Chromatin fragmentation was achieved by micrococcal nuclease treatment, and modified histones were immunoprecipitated using an H3K27me3 antibody (Cell signaling, Cat#9733) and ChIP Grade magnetic beads.
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| Growth protocol |
Epithelial cells were harvested from the mammary glands of 8-12-week-old MIC mice and grown as organoids. Roughly 10,000 mammary epithelial cells were cultured in 8-well chambers in the organotypic medium consisted of Epicult-B mouse medium (Stem Cell Technologies, 05610), knockout serum replacement (Gibco, 10828010), penicillin/streptomycin, 10 ng/mL EGF, 25 μg/mL insulin (Sigma, 10156), and 1 μg/mL hydrocorticone for 6 days to allow formation of acinar structures. Subsequently, doxycycline was added to growth media either in the presence or absence of drug treatments for 8 days, with media changes every 48hrs.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed using a SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling, Cat#9003) based on the manufacturer’s instructions.
The libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S/#E7103S), according to the manufacturer's instructions. Library construction protocol & strategy: (1) The DNA fragments are repaired, dA-tailed. (2) The DNA fragments with A tail are ligated to sequencing adaptors. (3) The final DNA library is obtained by size selection and PCR amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Data quality was assessed using FastQC
Sequence reads were trimmed using Trimmomatic as follows: (1) Discard the reads with low quality (proportion of low quality bases larger than 50%); (2) Discard the reads with N ratio (unsure base) larger than 15%; (3) Discard the reads with adaptor at the 5’-end; (4) Discard the reads without adaptor and inserted fragment at the 3’-end; (5) Trim the adapter sequence at the 3’-end; (6) Discard the reads whose length are less than 18nt after trimming.
Trimmed sequence reads were mapped to GrCM38/mm10 using BWA
Peaks were called using MACS2 software (Yong Zhang, Tao Liu et al., 2008) using a threshold q value of 0.05
BAM files were generated for each sample and visualized using IGV
Assembly: GrCM38/mm10
Supplementary files format and content: bigwig files
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| Submission date |
Jun 16, 2023 |
| Last update date |
Jul 14, 2023 |
| Contact name |
William Muller |
| E-mail(s) |
william.muller@mcgill.ca
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| Organization name |
McGill University
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| Department |
Biochemistry
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| Lab |
GCRC 507
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| Street address |
1160 Av des Pins O
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3A 1A3 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE235144 |
Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways (ChIP-Seq) |
| GSE235147 |
Ezh2 promotes mammary tumor initiation through the epigenetic regulation of the Wnt and mTORC1 signaling pathways |
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| Relations |
| BioSample |
SAMN35776858 |
| SRA |
SRX20709598 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7495933_Input2_D.bw |
145.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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