Method for the Enrichment of N⁶-Methyladenosine-Modified Cellular and HIV-1 RNA

N⁶-methyladenosine (m⁶A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m⁶A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4⁺ T-cells or HIV-1 envelope protein treatment upregulates m⁶A levels of cellular RNA. Changes in the m⁶A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m⁶A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m⁶A-modified RNA from HIV-1-infected primary CD4⁺ T-cells based on immunoprecipitation. The enriched RNA with m⁶A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.