GSM1716764: HOXB13 Chip-seq LNCaP; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
Sequenced DNA Library
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).