|
Status |
Public on Mar 24, 2016 |
Title |
MCF7_input_REP1 |
Sample type |
SRA |
|
|
Source name |
MCF7_input
|
Organism |
Homo sapiens |
Characteristics |
cell line: MCF7 tissue source: breast cell type: mammary gland/breast epithelial cell; metastatic site derived neoplasia type: adenocarcinoma atcc id: ATCC HTB-22 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.
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|
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 1500 |
|
|
Description |
MCF7 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
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|
|
Submission date |
Feb 10, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Jonathan AR Gordon |
E-mail(s) |
Jonathan.A.Gordon@uvm.edu
|
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 |
|
Relations |
BioSample |
SAMN04484752 |
SRA |
SRX1569798 |