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Sample GSM2058903 Query DataSets for GSM2058903
Status Public on Mar 24, 2016
Title MCF10A_input_REP1
Sample type SRA
Source name MCF10A_input
Organism Homo sapiens
Characteristics cell line: MCF10A
tissue source: breast
cell type: mammary gland/breast epithelial cell; luminal ductal cells
neoplasia type: fibrocystic disease
atcc id: ATCC CRL-10317
chip antibody: none (input DNA control)
Treatment protocol Cells were seeded at 1.5x10^6 cells per 100 mm dish and grown to 80% confluence. Cells were washed twice with phosphate buffered saline (PBS) and crosslinked with 0.8% formaldehyde (Thermo Scientific Cat no. 28908) in PBS for 10 minutes at room temperature with continuous shaking
Growth protocol MCF10a cells were grown in DMEM: F12 (Hyclone-SH30271 or SH30272 without phenol red), 5% (v/v) horse serum (Gibco #16050 lot #1075876) + 10ug/ml human insulin (Sigma I-1882)+ 20ng/ml recombinant hEGF (Peprotech AF-100-15) + 100ng/ml Cholera toxin (Sigma C-8052) + 0.5 ug/ml Hydrocortisone (Sigma H-0888) Pen/Strep (Life Technologies) and Glutamine (Life Technologies). MCF-7 cells and MDA-MB-231 were grown in DMEM: F12 (Hyclone-SH30271 or SH30272 without phenol red) + 10% (v/v) FBS (Atlanta Biologicals).
Extracted molecule genomic DNA
Extraction protocol Nuclei were isolated using a modification of the Dignam method (Dignam et al Nucleic Acids Res. 1983) and pellets were sonicated using a Covaris S-220 Ultrasonic Processor to obtain sheared chromatin ranging from 200-800 bp with an average peak size of 500 bp. For immunoprecipitation, 30 μg of sheared chromatin was incubated with 10 ug of anti-H3K4me3 (Abcam, ab1012 lot#GR80367-1) or H3K27me3 (Millipore -07-449 lot#1764447 or 2475696) overnight at 4°C and then incubated with 50 μl Protein-G Dynabeads (Life Technologies 10004D lot#123085320) for 4 h at 4°C.
Briefly, end-repair, A-tailing and paired-end adapter ligation were performed using 8 ug of ChIP-DNA at 150 pg/ul using the TruSeq ChIP sample preparation kit (Illumina cat#9235121 lot #15027084) following manufacture’s instructions. Excess adapters were removed by sequential Ampure XP (Beckman Coulter A63881 lot #1348300) purifications and recovered, ligated fragments were the amplified by PCR on a Biorad thermal cycler. Libraries were then run on a 2% agarose/TAE gel to select for fragments in the 350-400 bp range.
Barcoded libraries (Illumina TruSeq) were then loaded onto an Illumina HiSeq 1500 and single-end 100-base (SE100) sequencing was performed at the Advanced Genome Technologies Core Massively Parallel Sequencing Facility at UVM.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
Description MCF10A SAMPLE 5
Data processing Remove adapter reads (Cutadapt v1.6)
Trim low quality base calls from both ends. Min score >= 20, window of 10 step size of 1. (FASTQ Quality Trimmer 1.0.0)
Concatenate replicates.
Align reads with STAR v2.4, splicing disabled with '--alignIntronMax 1'.
Call peaks and generate .bdg with MACS2, p-value e-5. (MACS2 v2.1.0.20140616)
Compare treatment bedgraph to input with MACS2 bdgcmp in logFE mode.
Convert logFE.bdg to bigwig with UCSC's bedGraphToBigWig v4
Genome_build: hg38
Supplementary_files_format_and_content: bigwig files of log fold enrichment over input from bdgcmp function of MACS2
Submission date Feb 10, 2016
Last update date May 15, 2019
Contact name Jonathan AR Gordon
Organization name University of Vermont
Department Biochemistry
Street address 89 Beaumont Ave Given E209
City Burlington
State/province VT
ZIP/Postal code 05405
Country USA
Platform ID GPL18460
Series (1)
GSE77772 Bivalency - Histone H3 Lysine4 and Histone H3 Lysine27 Methylation Dynamics
BioSample SAMN04484746
SRA SRX1569792

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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