Total RNA from HEK293T cells were extracted using TRIzol reagent (Invitrogen, #15596018) 20 μg of total RNA extracted from HEK293T cells were subjected to poly(A)+ RNA selection using Dynabeads mRNA DIRECT purification kit (Invitrogen, #61011) following the manufacturer's instructions. Approximately 500 ng of the resulting poly(A)+ RNA, along with 25 ng of SIRV-Set1-E2 RNA, were pooled in one tube as input for direct RNA library generation. The libraries were made by following the standard SQK-RNA002 protocol with the optional reverse transcription step included.