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That's all up to you how you want to handle it.
But generally speaking when I do it

Layers:

Players
Enemies
Colliders

Ground
Foregrounds

Hello there! That's an interesting question. These tiles may work with as few as two layers (a terrain / structure layer, and an "overlay" layer for elements such as treetops and decorations with transparency). By following this strict limitation, you may be able to obtain a real retro-styled GBC aesthetic. Of course, on top of these two layers, a third one is dedicated to characters and animation.

I usually work with two or three layers as well + character/event layer, as this allows me slightly more freedom with overlaying tiles and transparent ones without making too many specific junction tiles!