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| Status |
Public on Jun 09, 2024 |
| Title |
K27_TET1 |
| Sample type |
SRA |
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| Source name |
MCF7
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: Breast cancer
chip antibody: H3k27ac
treatment: Tet1 knockdown
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| Treatment protocol |
TET1, and TET2 genes targeting siRNA were purchased from Santa Cruz Technologies, USA. Breast cancer cells were cultured for 16-18h at 75-80% confluency and incubated in serum-free DMEM for 3 h before transfection. The optimal concentration of siRNA was added to the 1.5 mL tube along with the required volume of serum-free medium and INTERFERin (Polyplus Transfection, SA, France). The siRNA-INTERFERin complex was incubated at room temperature (RT) for 20 min. The cells were replaced with a complete DMEM medium and the siRNA-INTERFERin complex was added. The cells were incubated for 24 h, and 48 h after transfection required for the experimental protocol. Scrambled siRNA was used as control siRNA in all experiments
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| Growth protocol |
MCF-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose and sodium bicarbonate contents (HiMedia, India), 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA), and 1X antibiotic solution (HiMedia, India). Cells maintained in the humidified incubators at 37° C supplemented with 5% CO2. Cells were washed with 1X Phosphate buffered saline (HiMedia, India) under confluency and detached using 0.25% Trypsin-EDTA solution (HiMedia, India). The desired seeding density of the cells was followed based on the standard seeding density chart (Thermo Fischer, USA), and the cells were counted using an automated cell counter Countess II FL (Life Technologies, Thermo Fischer, USA) with 0.4% trypan blue. All the cell lines were routinely checked for mycoplasma contamination using the Lookout® Mycoplasma PCR detection kit (Cat.no: MP0035, Sigma-Aldrich, USA).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
BC cells were harvested and washed with 1X PBS. Approximately 1X106 cells were used per ChIP reaction. The siRNA transfected and the control cells were collected and crosslinked with 1% v/v formaldehyde followed by 0.125M glycine treatment. The cells were washed with 1XPBS containing 1mM phenyl methyl sulfonate (PMSF) (Sigma) and 1X protease inhibitor cocktail (Sigma). The cell pellets were further processed for nuclei preparation. Chromatin shearing was performed by sonication in 15-sec pulse ON and 45-sec pulse OFF for 15 cycles to get the sheared chromatin fragments in the size range of 200-800bp. The size-selected sheared chromatin was further immunoprecipitated with H3K4me1antibody (Invitrogen, Thermo Scientific, USA) and H3K27ac antibody (Invitrogen, Thermo Scientific, USA). The magnetic protein A beads were used to collect the chromatin immunoprecipitated complex. Other clean-up and purification steps were performed per the manufacturer’s instruction (Zymo Spin ChIP kit, Zymo Research, USA).
The ChIP input DNA was size verified with tape station profiling followed by low-input ChIP library preparation.
ChIP-sequencing 150*2 PE chemistry
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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 |
Raw reads obtained from the sequencing were quality checked with FASTQC and the reads of low quality lesser than 30 phred score were discarded. Adapter content were trimmed with cutadapt.
The reads were aligned against the hg19 human reference genome using the BWA-mem algorithm and obtained more than 95% effective alignment.
The bam files obtained were sorted using samtools sort and then converted it into bigwig files with 20bp bin size for visualization.
The aligned reads were processed with MACS2 peak caller with p-value of 0.05.
Assembly: hg19
Supplementary files format and content: narrow peak file
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| Submission date |
Nov 02, 2023 |
| Last update date |
Jun 09, 2024 |
| Contact name |
Samson Mani |
| E-mail(s) |
samsonn.m@gmail.com
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| Phone |
6381741841
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| Organization name |
Cancer Institute (WIA)
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| Department |
Molecular Oncology
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| Street address |
38, Sardar Patel Road
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| City |
Chennai |
| State/province |
Tamilnadu |
| ZIP/Postal code |
600036 |
| Country |
India |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE246900 |
TET-mediated DNA demethylation influences histone enhancers H3K4me1 and H3K27ac in breast cancer |
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| Relations |
| BioSample |
SAMN38082072 |
| SRA |
SRX22341853 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7880519_T1_K27_peaks.narrowPeak.gz |
13.0 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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