|
| Status |
Public on May 15, 2011 |
| Title |
LNCaP-H3K4me2-dht-siCTRL-Mnase-ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Prostate cancer cell line (LNCaP)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
sirna transfection: siCTRL (1027280)
agent: DHT
mnase digestion: yes
chip antibody: H3K4me2
chip antibody vendor: Upstate
chip antibody catalog#: 07-030
transgenes: none
|
| Treatment protocol |
LNCaP cells were cultured in RPMI 1640 supplemented with 10% FBS. Control (1027280) and the specific siRNA against FOXA1 (M-010319) were purchased from Qiagen or Dharmacon. One day prior to transfection, LNCaP cells were seeded in RPMI 1640 medium. Six hours after transfection with Lipofectamine 2000 (Invitrogen), cells were washed twice with PBS and then maintained in hormone-deprived phenol-free RPMI 1640 media. Cells were then cultured for 96 hours following transfection and then treated with DHT or vehicle for 4 hrs.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After formaldehyde fixation, the LNCaP cells were resuspended in digestion buffer with 50U MNase and incubated at 37 degree for 8 min, and then the reaction was stopped by adding EDTA. The digested cells should generate mainly mononucleosomes with minor fraction of dinucleosomes. The samples were then sonicated and subjected to same library preparation protocol as the other ChIP-seq samples.
Chromatin immunoprecipitation was performed using ~10 million cell crosslinked with 1% formaldehyde at room temperature for 15 min. After sonication, the soluble chromatin was incubated with 1-5ug of antibody. Specific immunocomplexes were precipitated with Protein A or G beads (Sigma-Aldrich). Complexes were washed and the DNA was extracted and purified by QIAquick Spin columns (Qiagen). For ChIP-seq, extracted DNA was ligated to specific adaptors followed by deep sequencing in the Solexa GAII system according to manufacturer's instruction (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against H3K4me2 mononucleosomes in LNCaP cells treated with control siRNA and with DHT for 4 hrs
|
| Data processing |
Alignment: Reads were truncated to 25 bp (ChIP-Seq) or 32 bp (RNA, GRO-Seq) and aligned to the human hg18 genome (NCBI Build 36) using Bowtie. Only reads that mapped to a single, unique position were used for downstream analysis.
Data analysis was performed using HOMER, a software suite for high-throughput sequencing analysis and created in part to support this study (http://biowhat.ucsd.edu/homer/). Each sequencing experiment was normalized to a total of 10 million uniquely mapped tags by adjusting the number of tags at each position in the genome to the correct fractional amount given the total tags mapped. UCSC Genome Browser bedGraph files were created by calculating ChIP-Fragment pileups given a fragment size of ~150 bp. GRO-Seq bedGraph files are strand specific and contain pileups assuming a RNA fragment size of ~75 bp. Focal peaks (~200 bp) were found for transcription factors/co-factors, and broad, variable lengthed regions of enrichment were found for histone modifications using the default settings in HOMER. GRO-Seq data was initially pooled across all samples to identify contigous regions of nascent RNA enrichment to identify transcription units without prior knowledge of genes.
|
| |
|
| Submission date |
Mar 08, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE27823 |
Reprogramming Transcriptional Responses through Functionally-Distinct Classes of Enhancers in Prostate Cancer Cells [ChIP-Seq, Gro-Seq] |
| GSE27824 |
Reprogramming Transcriptional Responses through Functionally-Distinct Classes of Enhancers in Prostate Cancer Cells |
|
| Relations |
| SRA |
SRX047070 |
| BioSample |
SAMN00217261 |
