GSM2650748: Stat3 Input2; Mus musculus; ChIP-Seq
Formalin (Sigma) was added to a final concentration of 1% for 10m to crosslink DNA and attached proteins. Crosslinking was halted by addition of 2.5M Glycine in water to a final concentration of .125M and left to incubated at room temperature for 5m. Dishes were then washed twice in PBS, scraped, and pelleted by centrifugation at 1,200 RPM for 5m at 4oC. An extra dish was trypsinized and counted to determine OPC number per plate. Media was removed by suction and pellets were resuspended in cell lysis buffer (85mM KCl, 0.5% NP40, in 5mM HEPES, pH 8) with 200X protease inhibitor cocktail (Cell Signaling) and PMSF (Sigma) in isopropyl alcohol to a final concentration of 1mM being added fresh. Lysed cells were pooled together to a calculated density of 60 million cells in 600μl cell lysis buffer. Lysed cells were then sonicated at 4oC in a Bioruptor plus (Diagenode, Denville, NJ, USA) for 25 cycles (30s on, 30s rest) on high. Resulting suspension was then centrifuged at 14,000RPM at room temperature for 15m to pellet cell debris. Supernatants were transferred to 1.5ml tubes and 3μl of each were removed for DNA extraction (SimpleChIP Chromatin IP Kit, Cell Signaling). Extracted DNA was assessed for fragment size on an Agilent 2100 Bioanalyzer and DNA concentration on a QUBIT 3.0 Fluorometer (Life Technologies, Norwalk, CT, USA). Upon confirmation of sufficient fragment distribution (>10% between 130 and 230bp), 1% of volume from each tube was taken and saved as input. Supernatants were aliquoted proportionally to 30 million cells per tube and anti-Stat3(y705) (Cell Signaling, 1:100) or normal rabbit IgG (Cell Signaling 1:100) were added to each. Tubes were incubated overnight at 4oC with rotation. The next day 30μl of ChIP Grade Protein G Magnetic Beads (Cell Signaling) was added to each reaction for 2h at 4oC with rotation. Washing of immunoprecipated chromatin, elution of chromatin, and DNA purification of immunoprecipitated samples and inputs using spin columns were carried out according to manufacturer’s protocol (SimpleChIP Chromatin IP Kit, Cell Signaling). 10ng of each ChIP and input sample were then used for library construction for Illumina sequencing following manufacturer’s protocol (NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina, NEBNext Multiplex Oligos for Illumina (Index Primers Set 1), New England BioLabs, Ipswich, MA, USA).
GEO Accession
GSM2650748