Purification of Nascent, Chromatin-Associated RNA Enriched for RNA Modifications

RNAs that are being synthesized and remain tethered to chromatin during transcription are key participants in co-transcriptional gene regulation. They contribute to essential processes such as splicing, transcription elongation, and chromatin structure shaping. Co- and post-transcriptional RNA modifications have emerged as critical regulators of RNA metabolism and may modulate the function of chromatin-associated RNAs, including their interactions with chromatin. Here, we describe a method to isolate nascent, chromatin-associated RNA enriched for specific RNA modifications, using a combination of metabolic labeling, cell fractionation, and immunoprecipitation. This approach involves labeling of newly transcribed RNA with nucleotide analogs, followed by subcellular fractionation to enrich for chromatin-bound RNA species. Subsequently, immunoprecipitation with RNA modification-specific antibodies is used to selectively purify RNA bearing specific epitranscriptomic marks. We detail each component of the protocol, which can be adapted to various mammalian cell types and experimental conditions. The nature of this workflow enables investigation of RNA modifications on nascent, chromatin-associated transcripts under physiological or perturbed conditions, including various stimuli, genetic mutants, or disease-relevant cell lines.