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| Status |
Public on Nov 16, 2022 |
| Title |
Menin_ChIP_3T3_MI-503 |
| Sample type |
SRA |
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| Source name |
NIH-3T3 fibroblasts
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| Organism |
Mus musculus |
| Characteristics |
cell line: NIH-3T3
cell type: Fibroblasts
chip antibody: Menin
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| Growth protocol |
Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal calf serum (ATCC) and Penicillin-Streptomycin. Cells were harvested four days after being treated with Menin-MLL inhibitor in this same media composition.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linking ChIP in mouse NIH-3T3 cells was performed with 10-20x107 cells per immunoprecipitation. After drug (or vehicle) treatment, cells were collected, washed once with ice-cold PBS, and flash-frozen. Cells were resuspended in ice-cold PBS and cross-linked using 1% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 5 minutes at room temperature with gentle rotation. Unreacted PFA was quenched with glycine (final concentration 125mM) for 5 minutes at room temperature with gentle rotation. Cells were washed once with ice-cold PBS and pelleted by centrifugation (800g for 5 minutes). To obtain a soluble chromatin extract, cells were resuspended in 1mL of LB1 (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1X complete protease inhibitor cocktail) and incubated at 4°C for 10 minutes while rotating. Samples were centrifuged (1400g for 5 minutes), resuspended in 1mL of LB2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 1X complete protease inhibitor cocktail), and incubated at 4°C for 10 minutes while rotating. Finally, samples were centrifuged (1400g for 5 minutes) and resuspended in 1mL of LB3 (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N- Lauroylsarcosine, 1X complete protease inhibitor cocktail). Samples were homogenized by passing 7-8 times through a 28-gauge needle and Triton X-100 was added to a final concentration of 1%. Chromatin extracts were sonicated for 14 minutes using a Covaris E220 focused ultrasonicator. Lysates were centrifuged at maximum speed for 10 minutes at 4°C and 5% of supernatant was saved as input DNA. Beads were prepared by incubating in 0.5% BSA in PBS and antibodies overnight (100μL of Dynabeads Protein A or Protein G (Invitrogen) plus 20μL of antibody).
NEBNext Ultra II DNA
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
ChIP-seq reads are aligned using Rsubread’s align method and predicted fragment lengths calculated by the ChIPQC R Bioconductor package. Normalized, fragment extended signal bigWigs are created using the rtracklayer R Bioconductor package. Peak calls are made in MACS2 software.
Genome_build: mm10/hg38
Supplementary_files_format_and_content: bigwig files normalized to read depth
Supplementary_files_format_and_content: narrowPeak files generated by MACS2
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| Submission date |
Oct 27, 2021 |
| Last update date |
Nov 16, 2022 |
| Contact name |
Douglas Barrows |
| E-mail(s) |
dbarrows@rockefeller.edu
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| Phone |
7749940981
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| Organization name |
Rockefeller University
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| Street address |
1230 York Ave
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE186695 |
A molecular switch between mammalian MLL complexes dictates response to Menin-MLL inhibition [ChIP-seq] |
| GSE186711 |
A molecular switch between mammalian MLL complexes dictates response to Menin-MLL inhibition |
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| Relations |
| BioSample |
SAMN22604511 |
| SRA |
SRX12799813 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5659066_Sorted_3T3_Ctrl_Menin_ChIP_MI_503Normalised.bw |
27.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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