The Spider's Rune
is located in the Slumbering Sanctuary
. You access the Slumbering Sanctuary
after you beat the Conjunctivitis boss in the Insufferable Crypt
. The Ram Rune
is required to get there.
Once you have the Ram Rune, you need to take this path:
Prisoners' Quarters > Toxic Sewers > Ancient Sewers > Insufferable Crypt > Slumbering Sanctuary
I found this map to be very helpful.