Hi there!
I first use photoshop for all my pixel needs. When I create a new project, I select new, and make sure I select the transparent background option.
After I'm done with my pixels, and I want to animate my art, I use Image Ready to create make it move, or change it's color.
When I go straight to image ready, it saves all my pixel work, and transfers it immediately to Image Ready, with the transparent background. I then save as optimized, and it save it as a .gif.
Hope that helps.