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Sample GSM1494437 Query DataSets for GSM1494437
Status Public on Feb 12, 2015
Title HuRef_H3_B_(20140605_2,10)
Sample type SRA
Source name Immunoprecipitated DNA
Organism Homo sapiens
Characteristics cell: nuclei
cell type: Lymphoblastoid
cell line: HuRef
chip antibody: Anti-H3 (Abcam cat #Ab1791)
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 (Abcam anti-H3 Ab1791) 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 KAPA polymerase.
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina HiSeq 2500
Description HuRef native H3 ChIP using KAPA polymerase.
Data processing Paired reads were merged using SeqPrep ( with parameters -q 30 (quality) and -L 25 (minimum merged pair length). SeqPrep also removed adaptors and low-quality reads.
Submission date Aug 29, 2014
Last update date May 15, 2019
Contact name Jorja Henikoff
Phone 206-667-4850
Organization name Fred Hutchinson Cancer Research Center
Department Basic Sciences
Lab Henikoff
Street address 1100 Fairview AV N, A1-162
City Seattle
State/province WA
ZIP/Postal code 98109-1024
Country USA
Platform ID GPL16791
Series (1)
GSE60951 A unique chromatin complex occupies young alpha-satellite arrays of human centromeres
BioSample SAMN03013863
SRA SRX690215

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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