P-GSE40140-5 - P-GSE40140-5
nucleic acid extraction protocol
Protocols describing materials and methods for ChIP have previously been described in detail by Kaufmann et al. (2010). Briefly, 1.5 g of 14-day-old seedlings was chemically crosslinked by the addition of 1% formaldehyde for 15 minutes at room temperature. The fixed seedlings were rinsed five times with MC buffer (10 mM sodium phosphate, pH 7, 50 mM NaCl and 0.1 M sucrose) and frozen in liquid nitrogen. The frozen samples were homogenized, filtered, centrifuged and washed to isolate nuclei. The nuclear pellets were resuspended, lysed and sonicated to shear crosslinked DNA. The sonication was carried out with a Bioruptor UCD-200 (Diagenode, Liège, Belgium) until the average size of sheared DNA was approximately 500 bp. The sonicated chromatin was pre-cleared with protein-A agarose beads for 1 hour and immunoprecipitated with 2 μg of anti-c-myc antibody for 2 hours followed by addition of protein-A agarose beads for 3 hours. The beads were washed five times with IP buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10 μM ZnSO4, 1% Triton X-100 and 0.05% SDS). The bound IP complexes were eluted with elution buffer (0.1 M glycine, 0.5 M NaCl and 0.05% Tween-20, pH 2.8) and neutralized with 1 M Tris, pH 9.0. The eluantes were digested with proteinase K overnight at 37 oC to remove proteins followed by incubation at 65 oC for at least 6 hours to reverse crosslinking. The reverse crosslinked DNA was then purified by extraction with phenol:chloroform:isoamyl alcohol and ethanol precipitation in the presence of glycogen. The DNA pellets were washed with 70 % of ethanol, air dried and resuspended in TE 8.0.