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| Status |
Public on May 24, 2016 |
| Title |
HT10801b_CenpA_CenpB_X_(20151015_10) |
| Sample type |
SRA |
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| Source name |
Sequential immunoprecipitated DNA
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| Organism |
Homo sapiens |
| Characteristics |
cell type: fibrosarcoma
cell line: HT1080-1b
chip antibodies: Anti-FLAG M2 magnetic beads (Signa Cat # M8823) and Anti-CENP-B (Abcam Cat #Ab25734)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The HT1080-1b fibrosarcoma cell line was grown in DMEM media using standard protocols. Antibodies used in this study were purchased from Abcam (Cambridge MA). Primary cross-linking ChIP was performed as described previously (PMID: 24737864) with few modifications. Briefly, for each IP 200 million cells were fixed at a density equal to 2 million/ml using formaldehyde to a final concentration of 1%. Cross-linking was quenched with 125mM glycine. Chromatin was released from cells by incubating in lysis buffer (1% SDS, 10mM EDTA and 50 mM Tris-HCl (pH 8.1)) on ice for 10 min. The resulting chromatin solution was diluted 10 times with IP dilution buffer (1% Triton X-100, 2mM EDTA, 150 mM NaCl and 20mM Tris-HCl (pH8.1)) and digested with Micrococcal nuclease (MNase, Sigma, Cat # N3755) at 37°C for 45 min. MNase digested chromatin was sonicated as described before ( PMID: 24737864)and solubilized chromatin was precleared using protein A sepharose beads for 30 minutes at 4 °C. The precleared chromatin was incubated with Anti-FLAG-M2 magnetic beads (Sigma Cat # M8823) for 4 hrs at 4 °C . The FLAG-M2 magnetic beads were washed once with buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), and 150 mM NaCl), four times with buffer II 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 150 mM NaCl), 2 times with buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and once with 1X TE. Protein complexes were eluted from magnetic beads using FLAG peptide in HE buffer (10 mM HEPES, 0.25 mM EDTA, 0.5 mM PMSF). The eluent was subjected to subsequent ChIP reaction with Anti-CENP-B (Abcam, Cat# Ab25734)antibodies.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 |
HT1080-1b cross-linked CENP-A/CENP-B Sequential ChIP
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| Data processing |
250bp single end reads were trimmed with trim-galore version 0.3.7 with parameters --quality 20 --adapter AGATCGGAAGAGC --stringency 3 --phred33 --length 25 and then aligned with various human alpha-satellite sequences using BWA 0.7.10 aln and sampe functions saving up to 10 alignments per pair, otherwise with default options. Trimmed reads 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 |
SAMN04364776 |
| SRA |
SRX1500114 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1979827_HT10801b_CenpA_CenpB_X.Cen1-like.bed.gz |
90.6 Kb |
(ftp)(http) |
BED |
| GSM1979827_HT10801b_CenpA_CenpB_X.Cen13-like.bed.gz |
14.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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