|
Status |
Public on Nov 12, 2014 |
Title |
Input (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: none
|
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. For ATAC-seq, cells were harvested, nuclei were prepped,and transposase was added for 30 minutes at 30C. Sequencing libraries for ChIP-seq were constructed using the NEBNext Ultra kit as per manufacturer's recommended instructions. Sequencing libraries for ATAC-seq were constructed using custom Nextera-compatible primers, from Nextera-adapted DNA fragments
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|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2000 |
|
|
Data processing |
Raw fastq files were aligned to hg18/ncbi36 using Bowtie, allowing for only uniquely aligned reads to be reported. bowtie --chunkmbs 512 -m 1 --best <hg18_index> -q <file.fastq> <file.map> 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 BOWTIE -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' Genome_build: ncbi36/hg18 Supplementary_files_format_and_content: Peak BED files (3 column, from MACS) Supplementary_files_format_and_content: BedGraph files (from HOMER, for visualization in UCSC) Supplementary_files_format_and_content: FPKM values of gene expression from RNAseq data in tab-delimited text format
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|
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Submission date |
Jun 23, 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 |
GPL11154 |
Series (1) |
GSE58740 |
Chromatin dynamics of p53 binding sites in IMR90 |
|
Relations |
BioSample |
SAMN02870096 |
SRA |
SRX620749 |