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Sample GSM1649544

Query DataSets for GSM1649544

Status

Public on Feb 23, 2017

Title

IgG_ChIPSeq_Rep_1

Sample type

SRA

Source name

primary melanocytes

Organism

Homo sapiens

Characteristics

passage: under 10 passages
tissue: neonatal foreskin
antibody: Mouse IgG (Millipore, Billerica, MA)

Growth protocol

Primary melanocytes were regularly maintained in culture using complete growth medium consisting of Minimum Essential Medium Eagle (MCDB) 153 supplemented with 4% fetal bovine serum, insulin (5 ug/ml), alpha-tocopherol (1 ug/ml), 1% penicillin/streptomycin/amphotericin, human basic fibroblast growth factor (bFGF, 0.6 ng/ml), phorbol 12-myristate 13-acetate (PMA, 8 nM), and bovine pituitary extract (BPE, 13 ug/ml). For splitting primary melanocytes T-75 cm2 flasks were washed with an EDTA solution for two minutes, solution removed, and then 500 ul of 0.25% trypsin solution added. Cells were incubated with trypsin solution for two minutes, flasks were tapped to help detach the cells, and then trypsin solution was neutralized with 10 ml of media. In general, one T-75 cm2 flask was split into 4 flasks (2.5 ml cell suspension + 8 ml media). All ChIP-Seq experiments were conducted on primary cells that had not exceeded 10 passages.

Extracted molecule

genomic DNA

Extraction protocol

For chromatin immunoprecipitation (ChIP) experiments approximately 40 million human primary melanocytes were fixed at room temperature for 10 minutes with 1% formaldehyde diluted in cell culture media (v/v). Following fixation, formaldehyde was quenched with 1.25M glycine for 5 minutes at room temperature. Fixed/quenched cells were subsequently spun at 1,200 rpm for 5 minutes, washed with 1x PBS, and respun. Pelleted cells were then resuspended in 1mL cell lysis buffer (150mM NaCl, 10mM Hepes pH 7.4, 1.5mM MgCl2, 10 mM KCl, 0.5% NP-40, 0.5mM DTT, 1mM EDTA, plus protease inhibitors) and incubated on ice 10 minutes. Following cell lysis, nuclei were pelleted by spinning down at 5,000 rpm, 5 minutes at 4 degrees Celsius. Nuclei were then resuspended in 1 part nuclear lysis buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS plus protease inhibitors) and 2 parts ChIP-dilution buffer (150mM NaCl, 16.7mM Tris pH 7.5, 3.3mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% Na-Doc, plus protease inhibitors), and either sonicated on a Covaris (Woburn, MA, Model S220) or with a standard probe-tip sonicator (VirTis Virsonic 600). Sonication settings were determined empirically to generate fragments ranging from 200-500 bp. Following sonication samples were spun at 14,000 rpm for 15 minutes and supernatant saved for immunoprecipitation. For immunoprecipitation, dynabeads (Life Technologies, Grand Island, NY) were first washed 3x with ChIP-dilution buffer and subsequently incubated with equal amounts (5-10ug) of either anti-TFAP2A (3B5, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or Mouse IgG (Millipore, Billerica, MA) for 2 hrs up to overnight at 4 degrees Celsius. Following incubation antibody bound beads were washed 3x with ChIP-dilution buffer. Sonicated chromatin was then equally split between two tubes and brought to approximately 1 mL with ChIP-dilution buffer and used to resuspend antibody bound beads. Samples were incubated at 4 degrees Celsius overnight. Following overnight incubation antibody/bead/chromatin complexes were washed with a series of solutions 3x followed by elution with 100 uL elution buffer. Subsequent to elution crosslinks were reversed overnight at 65 degrees Celsius with 0.2 M NaCl. DNA was further cleaned using a Qiagen PCR purification kit as per manufacturer's instructions (Qiagen, Valencia, CA).
Post-ChIP a variety of quality control protocols were implemented before sequencing. Initially, real-time PCR was performed to confirm successful chromatin enrichment at known TFAP2A targets (and lack of enrichment at "off-targets"). Subsequently, total DNA enriched was quantified using a PicoGreen dsDNA assay kit (LifeTechnologies, Grand Island, NY) and shearing efficiency confirmed using a High Sensitivity DNA Analysis Kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) on the input sample. Libraries were then constructed using ABI's SOLiD ChIP-Seq kit with barcoding (rev. 08/06/2010), per manufacturer's instructions (Life Technologies, Grand Island, NY). Following library construction, samples were sequenced using the ABI SOLiD 3 platform, using a 50bp sequencing run.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

AB SOLiD System 3.0

Data processing

LifeScope™ Genomic Analysis Software was used for Basecalling and alignment to hg19 genome
Data processed with default specifications for each program
Data quality is assessed using Phantompeak tools as per the guidelines of the Encode project.
peaks were called using SPP R program with the FDR setting of 0.05
Genome_build: hg19
Supplementary_files_format_and_content: Bed file with Chip-seq peak regions and the columns represent; chrom, chromStart, chromEnd, name, score, strand, signalValue, pValue, qValue, peak

Submission date

Apr 03, 2015

Last update date

May 15, 2019

Contact name

Robert A. Cornell

E-mail(s)

robert-cornell@uiowa.edu

Organization name

University of Iowa

Department

Anatomy and Cell Biology