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We modulated the grafting density (σ) of a random copolymer brush of poly(styrene-r-methyl methacrylate) on substrates to probe its effect on the formation of perpendicularly aligned lamellae of polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA). Supported by coarse-grained simulation results, we hypothesized that an increase in σ will allow us to systematically tune the block copolymer interfacial interactions with the substrates from being preferential to one of the blocks to being neutral toward both blocks and will thereby facilitate enhanced regimes of perpendicularly aligned lamellae. We verified such a hypothesis by using a simple grafting-to approach to modify the substrates and characterized the thickness window for perpendicular lamellae as a function of brush thickness (or σ) on the grafted substrates using scanning force microscopy (SFM) images and grazing incidence small-angle X-ray scattering (GISAXS) measurements. The experimental results validated our hypothesis and suggested that the σ of random copolymer brushes can be used as an additional versatile parameter to modulate the interfacial interactions and the resulting alignment of block copolymer films. [ABSTRACT FROM AUTHOR]