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Sample GSM1494436

Query DataSets for GSM1494436

Status

Public on Feb 12, 2015

Title

HuRef_H3_A_(20140605_1,10)

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 Phusion polymerase.

Library strategy

MNase-Seq

Library source

genomic

Library selection

MNase

Instrument model

Illumina HiSeq 2500

Description

HuRef native H3 ChIP using Phusion polymerase.

Data processing

Paired 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.

Submission date

Aug 29, 2014

Last update date

May 15, 2019

Contact name

Jorja Henikoff

E-mail(s)

jorja@fhcrc.org

Phone

206-667-4850

Organization name

Fred Hutchinson Cancer Research Center