Ab1220

MM2d cells were harvested 4 days after subculture and fixed using ice-cold 1% formaldehyde in PBS and applying vacuum infiltration (three rounds of 6 min on/4 min off). The cross-linking was stopped by the addition of 0.125 M glycine, infiltrating for another 5 min. The grinded material was resuspended in Extraction Buffer (0.25 M sucrose, 10 mM Tris–HCl, pH 8.0, 10 mM MgCl₂, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail for plant cell extracts (Sigma)). Nuclei were pelleted by centrifugation, resuspended in Lysis Buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF and protease inhibitor cocktail) and disrupted by sonication in a Bioruptor Plus (Diagenode) for 30–45 cycles of 30 s on and 30 s off, at high power mode. One μg of soluble chromatin was employed per ChIP reaction, using the following antibodies: anti-H3K9me2 (Abcam ab1220, 3 μg), anti-H3K27me1 (Millipore 07-448, 1 μg), anti-total H3 (Abcam ab1791, 2 μg), or anti-rat IgG (Abcam ab6703, 2 μg) as a negative control. Immune complexes were recovered with 50 μl of protein G agarose beads (SCBT) and washed and eluted essentially as described (28 (link)).

MM2d cells were collected at 4 days after subculture and fixed in 4% paraformaldehyde in microtubules stabilizing buffer (MTSB; 50 mM PIPES, pH 6.9, 5 mM EGTA, 5 mM MgSO₄), for 10 min plus 5 min with vacuum infiltration. Cells were washed with MTSB, PBS and water and air-dried on superfrost plus slides (Thermo Scientific). Cells were re-fixed in 4% paraformaldehyde in MTSB for 30 min and washed with MTSB. Cell walls were partially digested with 20 mg/ml driselase (Sigma) in MTSB for 45 min at 37°C and the slides were washed with PBS. Membranes were permeabilized with 10% DMSO, 3% Igepal CA-630 in MTSB for 1 h. Non-specific sites were blocked in 3% BSA, 10% Horse Serum (HS) in PBS for 1 h at 37°C. H3K9me2 and H3K27me1 were detected with antibodies (Abcam ab1220 and Millipore 07-448, respectively) diluted 1:1000 in 1% BSA, 10% HS, 0.1% Tween-20 in PBS at 4°C overnight. Slides were washed with 3% BSA in PBS and incubated with donkey anti-mouse 555 and anti-rabbit 488 (A-31570 and A-21206 Thermo Scientific, respectively) diluted 1:500 in 1% BSA, 10% HS, 0.1% Tween-50 in PBS for 1 h. Following washes in 3% BSA in PBS, nuclei were counterstained with DAPI (Merck), washed with PBS and mounted in Mowiol 4-88 (Sigma). The localization of H3K9me2 and H3K27me1 in immunostained cells was analyzed by confocal microscopy (LSM710 Zeiss). Images were processed using Fiji.