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| Status |
Public on May 24, 2016 |
| Title |
HuRef_CenpT_3_(20150213_1_19) |
| 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-T (Abcam cat #Ab114120)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The HuRef lymphoblastoid cell line was grown in RPMI medium 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 degrees 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 cell/ml. Micrococcal nuclease (MNase, Sigma, Cat # N3755) was added to ~2.5 units/ml, and digestion was carried out at 37 degrees 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 degrees C on nutator. Soluble chromatin was collected by centrifuging the mixture at 12000rpm at 4 degrees 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 degrees C, and 15 mcg of antibody was added per ChIP sample and incubated overnight at 4 degrees C. Dynabeads were added to the samples and incubated at 4 degrees 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 as described (PMID:22184235). Solexa library construction was performed as described previously (PMID:22025700) and PCR-amplified using Phusion polymerase.
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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 CENP-T ChIP. Replicate with Abcam antibody.
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| Data processing |
Paired reads were aligned with various human BACs using BWA 0.7.10 aln and sampe functions saving up to 10 alignments per pair, otherwise with default options. 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 files (Cen1-like and Cen13-like).
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| Submission date |
Jun 12, 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 |
SAMN03771566 |
| SRA |
SRX1057955 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1709885_HuRef_CenpT_3.Cen1-like.bed.gz |
87.9 Kb |
(ftp)(http) |
BED |
| GSM1709885_HuRef_CenpT_3.Cen13-like.bed.gz |
42.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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