|
| Status |
Public on Oct 12, 2015 |
| Title |
AR Chip-seq_LHSAR_LacZ_2 |
| Sample type |
SRA |
| |
|
| Source name |
LHSAR LacZ cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LHSAR
antibodies: AR
|
| Treatment protocol |
LHSAR cells were plated 1 day prior to infection with appropriate lentivirus and medium was replaced 24 hours later with medium with R1881. Cells were harvested 48 hours after addition of R1881.
|
| Growth protocol |
LHSAR cells were grown in PrEGM medium. LNCaP cells were grown in RPMI supplemented with 10% fetal calf serum. Cells were passaged every 3-4 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fresh-frozen radical prostatectomy specimens were chosen from the Dana-Farber Cancer Institute/Harvard Cancer Center SPORE biobank and database. A genitourinary pathologist (M.L.) reviewed HE stained slides from each case and isolated areas enriched >70% for prostate tumor tissue or normal prostate epithelium. Using a 2mm2 core needle, approximately four cores were extracted from the areas circled on the slide. The frozen cores were pulverized using the Covaris CryoPrep system (Covaris, Woburn, MA). The tissue was then fixed using 1% formaldehyde buffer for 18 minutes and quenched with glycerine. Chromatin was sheared to 300–500 base pairs using the Covaris E220 ultra-sonicator. The resulting chromatin was incubated overnight with 6ug antibody to AR (N-20, Santa Cruz Biotechnology, Dallas, TX) bound to protein A and protein G beads (Life Technologies, Carlsbad, CA). A fraction of the sample was not exposed to antibody to be used as control (input). The samples were de-crosslinked, treated with RNase and proteinase K, and DNA was extracted. The samples were then re-sheared to 50-100 base pairs using the Covaris ultra-sonicator, and concentrations of the ChIP DNA were quantified by Qubit Fluorometer (Life Technologies). For cell line experiments, cells were fixed with 1 % formaldehyde for 10 minutes and quenched with glycine. Isolated chromatin from 1 x 10^7 fixed cells was sonicated to 200-300 bp and subjected to immunoprecipitation with the appropriate antibodies as described above.
DNA was amplified using ThruPLEX-FD kit (Rubicon Genomics).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.7
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie version 0.12.7 with default settings. Only uniquely aligned tags were retained
Peaks were called using MACS2 with the following settings: Q value <= 0.01, shiftsize = 100 for AR ChIP
Genome_build: hg19
Supplementary_files_format_and_content: Bigwig files were converted from wig files generated using unique reads from MACS2
|
| |
|
| Submission date |
Jun 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Fugen Li |
| E-mail(s) |
fugen_li@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Street address |
450 Brookline Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE56288 |
Androgen receptor programming in human tissue implicates HOXB13 in prostate pathogenesis [ChIP-Seq] |
| GSE70079 |
Androgen receptor programming in human tissue implicates HOXB13 in prostate pathogenesis |
|
| Relations |
| BioSample |
SAMN03783386 |
| SRA |
SRX1067071 |
