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| Status |
Public on Mar 29, 2016 |
| Title |
IgG ChIP-Seq |
| Sample type |
SRA |
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| Source name |
castration-resistant prostate cancer (CRPC) cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Prostate, CRPC
cell line: LNCaP-C4-2B cells
antibody: IgG Ab
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| Treatment protocol |
Cells were treated with IGF ligand to activate histone H4 phosphorylation at Tyr88
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| Growth protocol |
LNCaP-C4-2B cells were grown in DMEM+F12 media supplemented with 10% FBS
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with pY88-H4 or IgG antibody.
Fifty nanograms of immunoprecipitated DNA was fragmented to 300 base pairs using a Covaris M220 Focused-ultrasonicator (Covaris, Inc., Woburn, MA) and then used to generate sequencing libraries using the Kapa Hyper Prep Kit (Kapa Biosystems, Wilmington, MA). The size and quality of the library was evaluated using the Agilent BioAnalzyer, and the library was quantitated with the Kapa Library Quantification Kit.
Each enriched DNA library was then sequenced on an Illumina NextSeq 500 sequencer. The sequencing yield was very good, with over 90 million reads in each sample, of which 82.8 (IgF) and 77.9 million (IgG), respectively, mapped uniquely to the human genome GRCh37.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The 75-nt paired end sequence reads were mapped to the genome using the BWA-MEM algorithm. Alignment information for each read was stored in the output file *.bam. Only reads that mapped uniquely with proper pairing were used in the subsequent analysis.
Peak regions were called using the MACS2 software with the following options -f BAMPE -SPMR -q 0.01 -broad. The "BAMPE" option was used for calculating fragment lengths from the paired end reads, "SPMR" for normalizing read depths to number of fragments per million reads, "broad" for compositing broad regions from nearby peak regions, and the qvalue (FDR) cutoff was set to 0.01. A total of 376 peak regions were called between the IgF sample and the IgG control.
Using the HOMER (v4.7, 8-25-2014) program (http://homer.salk.edu/homer/) we found 15 de novo motifs in pY88-H4 ChIP data
Genome_build: GRCh37
Supplementary_files_format_and_content: SPMR bigwigs
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| Submission date |
Mar 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Nupam Mahajan |
| E-mail(s) |
nupam.mahajan@moffitt.org
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| Phone |
813 745 4078
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| Organization name |
Moffitt Cancer Center
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| Department |
Drug Discovery Program
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| Street address |
10902 Magnolia Dr
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| City |
Tampa |
| State/province |
FL |
| ZIP/Postal code |
33612 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE79658 |
ChIP-sequencing of prostate cancer cells |
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| Relations |
| BioSample |
SAMN04588718 |
| SRA |
SRX1667245 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2100524_C42B_IgG.bw |
313.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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