It doesn't have to be assassins or combat-related either. It's a public inn. Lots of people spend the night there...

• Wake them up with a fight breaking out down the hall or out in the streets.
• Wake them up with a drunk banging on their door looking for "Martha", who the drunk knows is in there. (And if the drunk gets inside the room, make 'em start hugging a chair and telling the PC(s) how much they love her. If the PCs get rowdy with the man, have him break down crying because Martha died in that room and he misses her badly. If they lock him out, he sits outside the room or outside the window crying all night.)
• Wake them up with ridiculously loud sex next door.
• Wake them up with noises on the roof or outside their window— an unusually loud owl or cats fighting, for example.
• Wake them up with inn staff who apologize for disturbing them, but the staff needs to search the room for an extremely important or valuable item left behind by a previous— very rich or powerful —occupant.

Not only do all of the above things prevent the players from getting the effects of the long rest, it plants the seed that they need to find their own base of operations that won't interfere with their rest, thus removing an excessive amount of gold they may be hauling around.