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| Status |
Public on May 26, 2015 |
| Title |
p300 |
| Sample type |
SRA |
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| Source name |
Kasumi-1_ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Kasumi-1
tissue origin: peripheral blood
cell type: myeloblast
passages: p25-30
chip antibody: p300
chip antibody vendor: Santa Cruz Biotechnology, Inc
chip antibody cat. #: SC-585
chip antibody lot #: L0611
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| Growth protocol |
Kasumi-1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI-1640 media supplemented with 20% FBS. Cells were expanded every 3-4 days in T75 tissue culture flasks. Kasumi-1 cells growing in log phase (1-1.5 million cells/ml) were fixed with 1% formaldehyde for 10 minutes at room temperature and subsequently quenched with 0.25 M glycine. Approximately 30 million cells were used for each ChIP-Seq library.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Kasumi-1 cells growing in log phase were fixed with 1% formaldehyde for 10 minutes at room temperature and subsequently quenched with 0.25 M glycine. After washes in PBS, cells were flash frozen in liquid nitrogen. Thawed pellets were resuspended in Buffer A (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and rotated at 4ºC for 10 minutes. Following centrifugation, pellets were resuspended in Buffer B (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA) and rotated at 4ºC for 10 minutes. Pellets from centrifuged samples were resuspended in Buffer C (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine). Samples were aliquoted, 3 ml per tube, and sonicated to a fragment size of 100-500 base pairs using a 3.2 mm sonication probe (QSonica, Newtown, CT). Sheared chromatin (from approximately 30 million cells) was immunoprecipitated with antibodies overnight. All ChIP experiments included either normal goat or rabbit IgG (EMD Millipore, Billerica, MA) as controls. Antibody-lysate complexes were mixed with Protein G Dynabeads (Life Technologies, Grand Island, NY) for 2 hours. For AML1, ETO, H3K4me3, and H3K27me3 immunoprecipitations, beads were washed once with IP buffer, 3 times with RIPA buffer (50 mM HEPES, 500 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.25% sodium deoxycholate), once with PBS, and once with TE buffer. For N-CoR and p300 pulldowns, beads were washed once with IP buffer, once with high salt buffer (2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), 1 time with RIPA buffer (50 mM HEPES, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.25% sodium deoxycholate), once with PBS, and once with TE buffer. Protein-DNA complexes were extracted from beads at 37ºC in elution buffer (10 mM EDTA, 50 mM Tris-HCl, 1% SDS). For reverse crosslinking, supernatants from centrifuged samples were rotated overnight at 60ºC. RNAse and proteinase K treated samples were extracted with phenol:chloroform:isoamyl alcohol. Precipitated DNA was resuspended in 10 mM Tris-HCl, quantified with a Qubit fluorometer (Life Technologies, Grand Island, NY).
The Illumina protocol (Illumina, Inc., San Diego, CA) for ChIP-seq library generation was used with slight modifications. Approximately 5-10 ng chromatin was end-repaired (EpiCentre Biotechnologies, Madison, WI). Material was then A-tailed and ligated with adapters for single end deep sequencing (Illumina, Inc., San Diego, CA). Adapter modified DNA was size-selected, 300-400 base pair (bp) range, and then amplified using the Phusion polymerase (New England Biolabs, Ipswich, MA). Amplified ChIP libraries were size selected and sequenced on an Illumina GAIIx Genome Analyzer (Illumina, Inc., San Diego, CA) at the UMass Medical School Deep Sequencing Core Facility (Worcester, MA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Base calls and sequence reads were generated by Illumina CASAVA software (version 1.6, Illumina).
Reads were mapped to the human genome (GRCh37, hg19) using bowtie (version 0.12.8) with up to two mismatches allowed.
Peak calling was performed on pooled replicates using MACS (version 2.0.10.20131216) with default settings and retaining peaks with a p < 10^{-20} threshold.
Genome_build: hg19
Supplementary_files_format_and_content: Bed files correspond to summits (+/- 50 bp) for peaks with p < 10^{-20}.
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| Submission date |
Oct 30, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Troy W. Whitfield |
| Organization name |
University of Massachusetts Medical School
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| Street address |
55 Lake Ave. N.
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| City |
Worcester |
| ZIP/Postal code |
01655 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE62847 |
Genome-wide co-occupancy of AML1-ETO and N-CoR defines the t(8;21) AML signature in leukemic cells |
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| Relations |
| BioSample |
SAMN03153407 |
| SRA |
SRX747574 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1534444_p300.summits.bed.gz |
130.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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