GSM3579303: LNCaP Control siRNA 144hrs CTCF Rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
CTCF
Cell type
Cell type Class
Prostate
Cell type
LNCAP
Primary Tissue
Prostate
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
LNCaP Prostate Cancer Cells
cell line
LNCaP Prostate Cancer Cells
chip antibody
CTCF
treatment
20nM Non-Target siRNA pool
time
144hrs
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were scraped into ice-cold PBS containing protease inhibitors and collected by centrifugation at 500g for 5 minutes at 4˚C. Pellet was resuspended in ice-cold PBS and fixed with 1% formaldehyde for 15 minutes at room temperature. Fixation was quenched with addition of 125mM (final concentration) glycine for 5 minutes at room temperature. Fixed cells were centrifuged at 500g for 5 minutes at 4˚C and washed 2x with 10mls ice-cold PBS containing protease inhibitors. Pellet was resuspended in 1.5ml nuclei extraction buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA and 0.5% IGEPAL) per 10x106 cells and incubated on ice for 10 minutes. Suspension was dounced 10x with a tight dounce and centrifuged at 1000g for 5 minutes at 4˚C. Pellet was washed 1x with ice-cold PBS containing protease inhibitors and centrifuged. Pellet was resuspended in 1x sonication buffer (50mM Tris-HCl pH 8, 1% SDS, 10mM EDTA) and fragmented to ~300bp using a Branson probe Sonifier. Fragmented DNA was centrifuged and verified to be the correct size using gel electrophoresis. Pelleted DNA was resuspended in 1ml IP dilution buffer (16.7mM Tris-HCl pH 8, 0.01% SDS, 1% Triton X-100, 167mM NaCl, 1.2mM EDTA) per ChIP (each ChIP contains 5x106 cells) cells and pre-cleared with 30ul of Salmon Sperm DNA/Protein A agarose-50% Slurry (Millipore #16-157) per ChIP. 10ug of the following antibodies were added per ChIP and incubated overnight– CTCF (#07-729, Millipore), H3K4me3 (#39159, Active Motif), H3K27ac (#39133, Active Motif). Antibody/protein complexes were bound to Salmon Sperm DNA/Protein A agarose-50% Slurry. The samples were pelleted and washed once with low salt buffer (2mM EDTA, 0.1% SDS, 1% Triton X-100, 20mM Tris-HCl pH8.1, 150mM NaCl) once with high salt buffer 2mM EDTA, 0.1% SDS, 1% Triton X-100, 20mM Tris-HCl pH8.1, 500mM NaCl), once with LiCl buffer (1mM EDTA, 10mM Tris-HCl pH8.1, 250mM LiCl, 1% sodium deoxycholate, 1% IGEPAL) and twice with TE buffer (1mM EDTA, 10mM Tris HCl pH 8.1). Antibody/protein complexes were eluted off beads using elution buffer (1% SDS, 100mM sodium bicarbonate). Crosslinks were reversed with NaCl. Protein and RNA were degraded with Proteinase K and RNAse A, respectively. Libraries were prepared using TruSeq ChIP sample Preparation Kit (Catalog # IP-202-900).