|
| Status |
Public on Nov 12, 2014 |
| Title |
H3K4me2 ChIP (Nutlin-3a) |
| Sample type |
SRA |
| |
|
| Source name |
IMR90 fetal lung fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
treatment: Nutlin-3a (5uM final, 6hrs)
passages: 30-35
antibody: H3K4me2 (Millipore)
|
| Treatment protocol |
Proliferating IMR90 fibroblasts were treated with either DMSO or nutlin3a (5uM final) for 6hrs before harvesting.
|
| Growth protocol |
IMR90 fibroblasts were grown in DMEM with 10% FBS at 3% oxygen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, cells were crosslinked with formaldehyde (1% final) for 10min at room temperature, and harvested for sonication. Nuclei were extracted and chromatin was sheared to an average size of 200bp using a Diagenode Bioruptor.
For RNA-seq, cells were harvested and PolyA+ RNA was isolated using the NEBNext Ultra RNA-seq Isolation Module.
Sequencing libraries for ChIP-seq were constructed using the NEBNext Ultra kit as per manufacturer's recommended instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIP of H3K4me2 from Nutlin-treated IMR90.
|
| Data processing |
Raw fastq files were aligned to hg19/ncbi37 using Bowtie2, allowing for only uniquely aligned reads to be reported.
bowtie2 -k1 -N1
Significant Peaks were called using macs (v1.4) with input controls and default mfold
macs -t <ChIP_Bowtie_output.map> -n <Condition_Input.map> -s 50 -f SAM -g 3107677273
Only peaks with an F.D.R < 1% were used in the analysis.
HOMER was used to generate BedGraph files for visualization. First, HOMER-specifc TagDirectories were generated from bowtie output files (file.map) using the command 'makeTagDirectory <ChIP_TagDirectory> <ChIP_Bowtie_output.map> '
BedGraphs were then generated using the command 'makeUCSCfile <ChIP_TagDirectory> -o auto', and bigWigs were generated using bedGraph2bigWig.pl
TSS, Enhancers, and Protoenhancer BED files were processed using H3K4me1 and H3K4me3 overlap with p53_Nutlin_Peaks_hg19_FDR1.txt.
Genome_build: ncbi37/hg19
Supplementary_files_format_and_content: Peak BED files (3 column, from MACS)
Supplementary_files_format_and_content: BigWig files (from HOMER, for visualization in UCSC)
Supplementary_files_format_and_content: FPKM values of gene expression from RNA-seq data in tab-delimited text format.
|
| |
|
| Submission date |
Oct 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Morgan Sammons |
| Organization name |
University of Pennsylvania
|
| Street address |
3400 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19129 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE58740 |
Chromatin dynamics of p53 binding sites in IMR90 |
|
| Relations |
| BioSample |
SAMN03113076 |
| SRA |
SRX952177 |
