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| Status |
Public on May 24, 2016 |
| Title |
HuRef_CenpA_(20141219_1) |
| Sample type |
SRA |
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| Source name |
Immunoprecipitated DNA
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| Organism |
Homo sapiens |
| Characteristics |
cell part: nuclei
cell type: Lymphoblastoid
cell line: HuRef
chip antibody: Anti-CENP-A (Abcam anti-CENP-A Ab13939)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The HuRef lymphoblastoid cell line was grown in RPMI media using standard protocols. Antibodies used in this study were purchased from Abcam (Cambridge MA). Nuclei were prepared following a previously published protocol (PMID: 23644596). Briefly, for each IP 40-60 million cells were collected and washed with 1X PBS. Cells were re-suspended in ice-cold buffer I (0.32 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF and protease inhibitor) at a density equal to ~25 million cells/ml. An equal volume of ice-cold buffer I supplemented with 0.1% NP40 was added and samples were incubated on ice for 10 min. Nuclei solution was layered on ice-cold buffer III (1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF and protease inhibitor) and centrifuged at 10,000g for 20 min at 4 °C. The pellet was re-suspended in buffer A (0.34 M sucrose, 15 mM HEPES, pH 7.4, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 1 mM DTT, 0.1 mM PMSF and protease inhibitor, 3mM Cacl2) at density equal to ~ 32.5 million cells/ml.Micrococcal nuclease (MNase, Sigma, Cat # N3755) was added to ~2.5 units/ml, and digestion was carried out at 37°C for 5 min. Reactions were stopped by addition of EGTA to a final concentration of 20 mM and EDTA to 10 mM. The final NaCl concentration was adjusted to 215 mM and needle extraction was performed as described previously (PMID: 22184235) to enhance the solubility of kinetochore complex. The resulting solution was incubated overnight at 4 °C on nutator. Soluble chromatin was collected by centrifuging the mixture at 12000rpm at 4 °C for 8 min. Soluble chromatin was diluted 3X with 20 mM Tris, pH 8.0, 5 mM EDTA and 200 mM NaCl, and Triton-X was added at a final concentration of 0.1% (v/v). Next, the chromatin solution was pre-cleared using Protein A/G fast flow Sepharose beads for 20 min at 4 °C, and 15 µg of antibody (Abcam anti-CENP-A Ab13939) was added per ChIP sample and incubated overnight at 4 °C. Dynabeads were added to the samples and incubated at 4°C for 2 hrs. Immunoprecipitated complexes were washed 6 times with 50mM Phosphate buffer pH 7.4, 5mM EDTA, 200mM NaCl, and DNA was extracted from Dynabeads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
HuRef native ChIP CENP-A
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| Data processing |
Paired-end 25bp reads were merged using SeqPrep (https://github.com/jstjohn/SeqPrep) with parameters -q 30 (quality) and -L 25 (minimum merged pair length). SeqPrep also removed adaptors and low-quality reads.. Merged pairs were aligned with various human BACs using the aln/samse functions of BWA version 0.7.5 saving up to 10 alignments per read (-n 10), otherwise with default options. Merged pairs mapped to two consensus sequences representing 20 human centromere sequences are included as supplementary bed files (Cen1-like and Cen13-like).
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| Submission date |
Dec 23, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jorja Henikoff |
| E-mail(s) |
jorja@fhcrc.org
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| Phone |
206-667-4850
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| Organization name |
Fred Hutchinson Cancer Research Center
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| Department |
Basic Sciences
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| Lab |
Henikoff
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| Street address |
1100 Fairview AV N, A1-162
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109-1024 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE69839 |
CENP-T and CENP-C bridge adjacent CENP-A nucleosomes on young alpha-satellite dimers |
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| Relations |
| BioSample |
SAMN04364773 |
| SRA |
SRX1500111 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1979824_HuRef_CenpA.Cen1-like.bed.gz |
2.5 Mb |
(ftp)(http) |
BED |
| GSM1979824_HuRef_CenpA.Cen13-like.bed.gz |
239.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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