GSM3579312: IMR90_CTCF siRNA 144hrs CTCF Rep1; Homo sapiens; ChIP-Seq
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).
GEO Accession
GSM3579312