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| Status |
Public on Mar 02, 2012 |
| Title |
Osteoblasts_H3K27Me3_ChIP-seq |
| Sample type |
SRA |
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| Source name |
Osteoblasts derived from MSCs, H3K27Me3 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell identity: osteoblasts
cell type: differentiated cell
chip antibody: H3K27Me3
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| Treatment protocol |
No treatment.
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| Growth protocol |
MSCs derived from adult bone marrow were cultured and differentiated to osteoblasts as described by Jaiswal et al., 1997 (J Cell Biochem 64, 295-312 (PMID 9027589)). U2OS cells were cultured in McCoy's 5A medium supplemented with 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked in 3.7% formaldehyde, lysed and sonicated to obtain chromatin fragments in the range of 200 to 600 base pairs. Sonicated chromatin was incubated with primary antibody, H3K4Me3 (Millipore, 07-473) or H3K27Me3 (kind gift from Thomas Jenuwein/ Nicholas Shukeir), overnight at 4°C, followed by capturing primary antibodies by adding ProteinA/G magnetic beads (DynaBeads) and incubating at room temperature for 3-4 hrs. Captured chromatin was washed in low- and high-salt buffers, as well as TE. Chromatin fragments were subjected to simultaneous elution, de-crosslinking and Proteinase-K treatment at 65°C in elution buffer followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation.
Following elution, the immunoprecipitated DNA was end repaired and then purified with Agencourt AMPure magnetic beads. Fragments were then ligated with SOLiD System's P1 and P2 adaptors, followed by another purification step using Agencourt's AMPure magnetic beads, followed by nick translation and amplification using the SOLiD library PCR primers 1 and 2. After amplification, the samples were purified with the Agencourt AMPure magnetic beads once again and the final library was quantitated by performing qPCR and run through the Agilent Bioanalyzer for QC analysis. New England BioLabs NEBNext DNA Sample Prep Master Mix 3 was used for library construction.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
Osteoblasts-H3K27Me3
The anti-H3K27Me3 antibody was a kind gift from Thomas Jenuwein/Nicholas Shukeir.
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| Data processing |
Sequencing reads were aligned to genome build hg18 (NCBI 36) using Bioscope 1.2.1. The software guarantees finding all alignments between the first 25 base pairs of the read (seed) and the reference sequence with up to 2 mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and -2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported. Aligned reads were analyzed using Model-based Analysis of ChIP-Seq (MACS) to detect regions enriched for the histone marks (called peaks) with default settings. Input was used as background for peak identification.
Genome Build:
O2_hg18.gff3: hg18
O2vOinput_extend_peaks.bed: hg18
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| Submission date |
Feb 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
hari easwaran |
| Organization name |
Johns Hopkins University
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| Department |
Oncology
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| Lab |
Steve Baylin
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| Street address |
1650 Orleans Street, CRB1, Room 530
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| City |
BALTIMORE |
| State/province |
MD |
| ZIP/Postal code |
21209 |
| Country |
USA |
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| Platform ID |
GPL9442 |
| Series (2) |
| GSE35573 |
ChIP-seq analysis of H3K4Me3- and H3K27Me3-marked chromatin in mesenchymal stem cells (MSCs), osteoblasts derived from MSCs and the osteosarcoma cell line U2OS |
| GSE35576 |
Gene expression and ChIP-seq analyses of mesenchymal stem cells, osteoblasts and the U2OS osteosarcoma cell line |
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| Relations |
| SRA |
SRX118600 |
| BioSample |
SAMN00783654 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM871041_O2_hg18.gff3.gz |
2.4 Gb |
(ftp)(http) |
GFF3 |
| GSM871041_O2vOinput_extend_peaks.bed.gz |
536.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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