RNA sequencing library preparation workflows must balance data quality, efficiency, and flexibility to support diverse sample types in sequencing core laboratory settings. A critical determinant of library performance is adapter chemistry, which influences ligation efficiency, yield, and downstream sequencing quality. In this study, we evaluated the interoperability of the Watchmaker RNA Library Prep Kit with Polaris™ Depletion system and NEBNext® Multiplex Oligos, with emphasis on method development for streamlined, scalable workflows. Commercially available human and mouse total RNA were used as standardized inputs in replicates to assess reproducibility. Libraries were prepared following Watchmaker protocol and incorporating NEBNext® hairpin-loop adapters for double stranded cDNA ligation. Adapter concentrations were adjusted per Watchmaker recommendations, and ligation was followed by USER® Enzyme (a mix of uracil-DNA glycosylase and Endonuclease VIII) treatment to linearize the hairpin structure prior to PCR amplification. Unique dual indices (UDIs) were introduced during amplification to enable multiplexed sequencing. Across both sample types, this integrated workflow produced library yields exceeding protocol expectations, with low variability observed across replicates, indicating robust and reproducible performance. Sequencing on Illumina NextSeq platform, followed by analysis using an open-source pipeline, demonstrated high alignment rates, low rRNA content, and consistent duplication levels across replicates. Importantly, incorporation of NEBNext adapter chemistry did not compromise downstream sequencing metrics. Overall, this approach demonstrates that compatible adapter integration can maintain library quality while enabling flexible reagent use. These findings support scalable method development strategies in core facilities where workflow consistency, adaptability and high-throughput processing are essential for supporting diverse RNA-seq applications.