α 6ma

Immunofluorescence (IF) staining experiments followed previously described procedures (19 (link),42–44 (link)). For the single antibody staining, the primary antibodies were α -6mA (Synaptic Systems, 202003, 1:2000) (19 (link)) and α -HA (Cell Signaling, #3724, 1:200) and the secondary antibody was Goat anti-Rabbit IgG (H+L) (Invitrogen, A-21428, 1:4000). For the co-staining, cells were first incubated with α -HA (Covance, MMS-101P, 1:500, mouse) and its secondary antibody (goat anti-mouse IgG (H+L), Alexa Fluor 555, Invitrogen, A-21127, 1:4000). After crosslink by 3% PFA, α -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used. Digital images were collected using an Olympus BX43 microscope with an Olympus DP72 camera.

Cell fixation and immuofluoresence (IF) staining were performed as described previously (6 (link),39 (link),40 (link)). Cells were fixed during vegetative growth or at 10 h after mixing cells of two mating types, as indicated. For the 6mA staining, fixed cells were treated with RNase A (50 μg/ml or otherwise indicated) in phosphate-buffered saline (PBS) buffer for 2 h at 37°C. Non-treated cells were either incubated at 37°C or at room temperature (RT), as indicated. Cells were then incubated with 2N HCl for 20 min at RT and neutralized with Tris–HCl (100 mM, pH 8.5) for 10 min, prior to antibody incubation. The primary antibodies are α-6mA (Synaptic Systems, 202003, 1:2000) (5 (link),6 (link),10 (link)), α-HA (Cell Signaling, C29F4, 1:200), α-H3K9-acetylation (Abcam, ab10812, 1:500) and α-H3K14-acetylation (Abcam, ab52946, 1:500). The primary antibodies are incubated with cells at 4°C overnight (for α-6mA) or at RT for 2 h (for other antibodies). Cells were then incubated in the secondary antibody (Goat anti-Rabbit IgG (H+L), Invitrogen, A-21428, 1:4000) at RT for 1 h. Digital images were collected using a Leica DM2500 microscope with a Leica DFC450C camera.

Cells were fixed and permeabilized in 5 mL chilled acetone for 5 min before spreading onto polylysine-coated coverslips.⁴² (link) The 6mA staining experiment followed previously described procedures.¹⁹ (link) Antibody information: the primary antibody (α-6mA, Synaptic Systems, 202003, 1:2000); the secondary antibody (Goat anti-Rabbit IgG (H+L), Invitrogen, A-21428, 1:4000).