|
| Status |
Public on Feb 10, 2016 |
| Title |
MCF10A_H3K4ME3_R2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF10A_H3K4ME3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10A
cell type: mammary gland/breast epithelial cell
neoplasia type: luminal ductal cells
atcc id: fibrocystic disease; ATCC CRL-10317
chip antibody: H3K4me3
chip antibody vendor: Abcam
chip antibody cat. #: ab1012
chip antibody lot #: GR80367-1
|
| Treatment protocol |
Cells were seeded at 1.5x106 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 H3K4ac (Active Motif -39382 lot#29108001) 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 4
H3K4me3 ChIP
processed data file: MCF10A_H3K4ME3_logFE.bw
|
| 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 |
May 29, 2015 |
| 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 (2) |
| GSE69377 |
Histone H3 Lysine4 Acetylation-Methylation Dynamics Define Breast Cancer Subtypes [ChIP-seq] |
| GSE75169 |
Histone H3 Lysine4 Acetylation-Methylation Dynamics Define Breast Cancer Subtypes |
|
| Relations |
| BioSample |
SAMN03743990 |
| SRA |
SRX1043545 |
