Oct 03, 2023
m6A visualization/immunofluorescence of DamID
- Lucian Dipeso1,2,
- Emily Hatch1,2
- 1University of Washington;
- 2Fred Hutchinson Cancer Center
Protocol Citation: Lucian Dipeso, Emily Hatch 2023. m6A visualization/immunofluorescence of DamID. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdz6zlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2023
Last Modified: October 03, 2023
Protocol Integer ID: 88738
Keywords: DamID, m6A, Immunofluorescence
Funders Acknowledgement:
EM Hatch
Grant ID: R35GM124766-02
L DiPeso
Grant ID: T32CA009657
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.
Materials
100% MeOH
PBS
BSA
TritonX-100
Sodium azide
DpnI (NEB #r0176)
CutSmart buffer (NEB #b7204)
Rabbit anti-m6A (Synaptic #202 003)
Secondary antibody
Protocol
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