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| Status |
Public on May 15, 2024 |
| Title |
WT1,EEDKO,DoxycyclineRemoval, H3K27me3, Rep2 |
| Sample type |
SRA |
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| Source name |
HEK293FT
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293FT
cell type: Human embryonic kidney
chip antibody: H3K27me3 (CST, #9733)
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| Growth protocol |
DMEM, 10% fetal bovine serum, 2mM glutamine, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2. Doxycycline was added at a concentration of 1ug/ml
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells for ChIP were cultured in 15 cm plates (~10 million cells). Cell pellets were first washed with cold PBS, crosslinked at room temperature with 1% formaldehyde (ThermoFisher Scientific) for 8 min. Crosslinking reactions were quenched by addition of 125 mM glycine for 10 min. Cell were then resuspended in Swelling Buffer (25 mM Hepes pH 7.8, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT) followed by Dounce homogenization. Nuclei were pelleted by centrifugation and then resuspended in sonication buffer (0.1% SDS, 1 mM EDTA and 10 mM Tri-HCl pH 8.0). The nuclei were sonicated to shear chromatin into ∼200-500 bp fragments using a Covaris E220. Sonicated samples were diluted with ChIP dilution buffer (0.1% SDS, 1 mM EDTA and 10 mM Tri-HCl pH 8.0, 1% Triton X-100, 150 mM NaCl). Diluted samples were centrifuged at 13,000 rpm for 10 min. The supernatant was used for immunoprecipitation using antibodies and 25 μl protein A/G beads for 12-16 h at 4°C (see Table S9 for antibodies). For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif# 53083) and histone H2Av antibody (Active Motif# 61686) were added as spike-in controls. ChIP-Seq samples for Flag antibody do not have spike-in controls. The beads were washed twice with high salt wash buffer A (50 mM Hepes pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, and 0.1% SDS), twice with wash buffer B (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Sodium deoxycholate, 0.5% NP-40) and twice with 1X TE (10 mM Tris-HCl, 1mM EDTA). The bound chromatin fragments were eluted with elution buffer (50 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaHCO3,1% SDS) twice for 10 min each at 65°C. Eluted DNA-proteins complexes were incubated overnight at 65°C to reverse crosslinks. RNAase A followed by Proteinase K was then added to digest RNA and protein. DNA was further purified using phenol chloroform/PCR Purification Kit (QIAGEN)
For ChIP-seq, sequencing library was constructed using TruSeq DNA sample Prep Kits (Illumina) and adapter dimers were removed by 2% agarose and Tris-acetate-EDTA gel electrophoresis. Size selected and purified DNA libraries were sequenced on an Illumina NextSeq 500 machine (Bauer core facility at Harvard University) to obtain 75 bp single-end reads.
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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 were quality controlled with fqc (v0.11.5)
Pooled libraries were demultiplexed using fastx.
Reads were aligned to custom reference genome HG19 with a reporter inserted at Chr11 (near WT1reporter coordinates hg19 32460504-32463612) or Chr3 (near TFRC reporter coordinates hg19 195809796-195812888) and Drosophila (dm3) using bowtie2 with default parameters. Bam files were generated with samtools 1.3.1
The mapped reads for H3K27me3 ChIP were normalized to scale factor using Deeptools, and mapped reads for Flag ChIP were normalized to reads per genome content using Deeptools and saved as bw files and visualized in IGV.
Assembly: HG19 with a reporter inserted at Chr11 (near WT1reporter coordinates hg19 32460504-32463612) or Chr3 (near TFRC reporter coordinates hg19 195809796-195812888) and Drosophila (dm3)
Supplementary files format and content: bigWig
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| Submission date |
Jul 28, 2022 |
| Last update date |
May 15, 2024 |
| Contact name |
Danesh Moazed |
| Organization name |
Harvard Medical School
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| Department |
Cell Biology
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| Lab |
Moazed Lab
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| Street address |
240 longwood avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE209986 |
Heritable human Polycomb silencing is locus-dependent and requires H2AK119ub1 self-propagation |
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| Relations |
| BioSample |
SAMN30025606 |
| SRA |
SRX16716995 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6412285_WT1reporter_EED_KO_DoxycyclineRemoval_H3K27me3_Rep2.bw |
538.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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