The research associated with this dataset explored cross-species prion transmission and the impact of cofactor environment on strain adaptation. Utilizing Protein Misfolding Cyclic Amplification with beads (PMCAb), hamster strain 263K was propagated under normal or RNA-depleted conditions first in mouse and then hamster substrates. A variety of readaptation experiments were conducted including brain analysis of hamsters inoculated with different PMCAb-derived material. Dataset includes Western blot and comparative histopathological brain images.