GSM3591446: Q11 treated with M2 antibody; Rattus norvegicus; ChIP-Seq
Anti-FLAG chromatin-IP (ChIP) was performed as Magna ChIP protocol described (Millipore, Billerica, MA, USA). Target cells were crosslinked by adding of 1% (v/v) of formaldehyde to a cell monolayer in growth medium followed by incubation for 10 min at 37°C. The crosslinking reaction was stopped by addition of 11% of the suspension volume of an ice cold 1 M solution of glycine.Media was removed and cells were washed twice with cold 1 × PBS buffer. The cells were spun down immediately (4 min, 2000 g, 4°C) and resuspended in cell lysis buffer (Millipore, Billerica, MA, USA, 2 × 107 cells per 500 μl) with protease inhibitor and incubated for 15 min on ice with vortex briefly every 5 min. To shear the chromatin into pieces of a few hundred base pairs, the cell lysate was sonicated using a vibra cell sonifier (Thermo Fisher Scientific, Waltham, MA, USA). The immunoprecipitation reaction was set up by combining 50 μl sonicated cell supernatant 10-fold in ChIP dilution buffer, 10 μg anti-FLAG M2 antibody and 20 μl of Protein G Magnetic beads for one ChIP reaction. This reaction was incubated overnight at 4°C. The beads were washed five times with wash buffer and TE-buffer using a magnetic particle concentrator (Millipore, Billerica, MA, USA). The bound chromatin was eluted twice by addition of 50 μl of ChIP elution buffer to the beads followed by centrifugation. Candidate DNA sequences were subsequently recovered by reverse crosslink and proteinase K treatment. Extracted DNAs were analyzed by Agilent Bio-analyzer and submitted to University of Chicago Functional genomic facility for library preparation (TruSeq ChIP Library Preparation Kit, IP-202-1012) and sequencing (HiSEQ2500).