m6A visualization/immunofluorescence of DamID
Lucian Dipeso, Emily Hatch
Abstract
Used for visualizing Dam activity by immunofluorescence in mammalian cells. This protocol stains N-6 methyladenosine (m6A) modified DNA by immunofluorescence while preserving other epitopes, avoiding harsh denaturation steps like heat or chemical treatments.
Steps
Protocol
Fix cells in 100% MeOH for 10 min at -20 °C
Block, and RNase treat in 2 ug/mL RNase A in blocking buffer (3% BSA, 0.04% TritonX-100, 0.02% sodium azide in PBS) at 37 °C for 30 min
Wash cells 2x in PBS for 5 min at room temperature
Permeabilize cells in 2% TritonX-100 in PBS for 10 min at room temperature
Digest in 50 U/mL DpnI (NEB #r0176) in 1x CutSmart buffer for 30 min at 37 °C
Wash 3x in PBS for 5 min at room temperature
Incubate cells in rabbit-anti-m6A (Synaptic, #202 003) diluted 1:500 in PBS for 30 min at room temperature in a humidified chamber
Wash 3x in PBS for 5 min at room temperature
Incubate cells in anti-rabbit, AlexaFluor conjugated antibody in blocking buffer, diluted according to manufacturer's suggestion, for 30 min at room temperature in a humidified chamber in the dark
Proceed with further immunofluorescence steps and/or DNA staining and mount cells
