An alternative to running dual rail would be to move the 'ground' reference from the bottom of the non-inverting input resistor and make this, say your voltage rail /2 or 2.5V. Then you would have an output whose output was V1-V2+2.5. It's usual when using single-rail circuits to assume some kind of mid-point (such as half-rail) as a reference instead of ground. You will need to make that reference 'stiff' enough, depending on your resistor values, - can use capacitor and resistors if you need only AC subtraction, but otherwise, might need another amp, or some other way to make low-ish impedance. If source impedance is known, though, you can just choose some resistor dividers carefully.
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