|
| Status |
Public on Aug 18, 2017 |
| Title |
0 hr TNF-a H3K4me1 ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
SW480_0 hr TNF-α H3K4me1 ChIP-seq
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SW480
cell type: Human Colon Cancer Cell Line
chip antibody: anti-H3K4me1 (ab8895)
|
| Treatment protocol |
All cells were treated with 12.5 ng ml-1 TNF-α (R&D Systems) for 0 or 16 hr before harvesting for GRO-seq, ChIP-seq, and RNA-seq analyses.
|
| Growth protocol |
Human SW480 cells were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco) medium supplemented with 10% FBS. SW480 cells that stably and inducibly express short hairpins against LacZ or p53 were kindly provided by Xinbin Chen (UC Davis) and were grown in standard DMEM medium containing 1X penicillin and streptomycin (Gemini Bio-Products), 1.5 μg ml-1 puromycin (Sigma), and were induced with 1 μg ml-1 doxycycline (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For GRO-seq analyses, nuclei were prepared and used for run-on reactions using BrUTP. Following nascent RNA enrichment with anti-BrdU agarose beads (SCBT), RNA was extracted using TRIzol LS reagent and used for library preparation. For ChIP, lysates from sonicated nuclei were prepared and protein-DNA complexes were IP'd with corresponding antibodies. For RNA-seq, total RNA was extracted using TRIzol LS reagent and strand-specific libraries were prepared from 1 mg of RNA
ChIP-seq libraries were prepared according to Illumina's ChIP Truseq protocol. RNA-seq libraries were prepared following the dUTP second strand cDNA method using NEXTflex barcodes. GRO-seq libraries were prepared similarly. Please refer to the methods section for detailed protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
RNA-seq:
Sequenced reads were trimmed for adaptor sequences and mapped to human hg38 reference genome using STAR (Ultrafast Universal RNA-seq Aligner)
Gene expression levels were counted per gene model (not counting dfferentiated splices) and differential expression across samples were determined using edgeR.
Gene Ontology enrichment was performed using Metascape.
ChIP-seq:
Sequenced reads were mapped to the hg38 human reference genome using Bowtie2 software and default parameters.
Mapped reads were then processed to make tag directories using the HOMER module. PCR duplications were removed and only uniquely mapped reads were kept for further analyses.
Genome browser files for resulting reads were generated by using the makeUCSCfile module from HOMER.
Enrichment for p65 and histone modification deposition wer ecalled using findPeaks module from HOMER using preset parameters for transcription factors and histones, respectively and compared to the input samples.
For p53 R273H peak calling, p53 enriched regions were determined by HOMER (comparing to inputs). The resulting regions were split into subpeaks using PeakSplitter software.
For identification of mutp53 and p65 peak colocalization, p65 peaks were first divided into 2 groups with and without uninduced mutp53 binding by using the co-bound option of mergePeak module from HOMER.
Deeptools were used to generate heatmaps and de novo motif analyses were performed using HOMER.
GRO-seq:
Sequenced reads were mapped to the hg38 human reference genome using Bowtie2 software.
Uniquely mapped reads were kept and at most 3 reads at each unique genomic position.
GRO-seq reads were counted 1000 bp around the specified transcription factor peaks and normalized to 10 million total reads using default settings of annotatePeaks module of HOMER.
Genome_build: hg38
|
| |
|
| Submission date |
Aug 18, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Shannon M Lauberth |
| E-mail(s) |
[email protected]
|
| Organization name |
UCSD
|
| Department |
Biological Sciences
|
| Lab |
Lauberth Lab
|
| Street address |
9500 Gilman Drive #0322, Bonner Hall 3202
|
| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE102796 |
Mutant p53 Shapes the Enhancer Landscape of Cancer Cells in Response to Chronic Immune Signaling |
|
| Relations |
| BioSample |
SAMN07516116 |
| SRA |
SRX3102564 |
