|
| Status |
Public on Feb 08, 2019 |
| Title |
UBR7-shRNA-H3K4me3_ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
MCF10A Cell Line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10A
cell type: Human mammary epithelial cell line
chip-antibody: H3K4me3 Abcam/Millipore ab
|
| Treatment protocol |
shRNA plasmids for UBR7 with pLKO.1-puro backbone (Sigma Aldrich) were screened for efficient knock-down.Two out seven shRNAs were selected for subsequent experiments. Four micrograms of shRNA and packaging vectors were transfected as described previously (ref). Cells were selected using puromycin (10 μg / ml) (Sigma) for 3days.
|
| Growth protocol |
MCF10A cells were maintained in DMEM / Ham’s F12 supplemented with 5% horse serum (Gibco), EGF, insulin, hydrocortisone, cholera toxin (Sigma) and 1% antibiotic-antimycotic.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone-DNA complexes were isolated using antibodies described from Control and UBR7-shRNA sonicated nuclei.
Libraries for Illumina sequencing were generated following the New England BioLabs NEBNext Ultra DNA Library Prep Kit protocol. A total of 10x cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments ~200 to 500bp and quantified using the Qubit 2000 and Bioanalyzer 1000 before multiplexing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Raw fastq reads for all ChIP-seq experiments were processed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and aligned to the hg19 reference genome using Bowtie version 1.1.2 with the following criteria: -n 1 -m 1 --best --strata. These criteria preserved only the best reads that uniquely mapped to the genome with 1 or fewer mismatches. Duplicate reads were marked using SAMBLASTER before compression to BAM files. To directly compare Control and UBR7-shRNA ChIP-seq samples, uniquely mapped reads for each mark were normalized by total reads per condition, sorted and indexed using samtools version 0.1.19
Genome_build: hg19
Supplementary_files_format_and_content: Model based analysis of ChIP-seq (MACS) version 1.4.2 peak calling algorithm with a p-value threshold of 1e-7 to identify H2BK120ub enrichment over “input” background
Supplementary_files_format_and_content: deepTools version 1.5.11 was used to generate bigWig files by scaling the bam files to reads per kilobase per million (RPKM). Scaled bigWig files were then converted to bedGraph files using the bigWigToBedGraph tool.
|
| |
|
| Submission date |
Jan 18, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kunal Rai |
| E-mail(s) |
Krai@MDAnderson.org
|
| Phone |
713-792-6809
|
| Organization name |
MD Anderson Cancer Center
|
| Department |
Genomic Medicine
|
| Lab |
Rai Lab
|
| Street address |
1801 East Rd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77054 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE93757 |
UBR7 is a novel E3 ubiquitin ligase for H2BK120 and acts as a tumor-suppressor in breast cancer [ChIP-Seq] |
| GSE93759 |
UBR7 is a novel E3 ubiquitin ligase for H2BK120 and acts as a tumor-suppressor in breast cancer |
|
| Relations |
| BioSample |
SAMN06237765 |
| SRA |
SRX2499865 |
