Lentiviral RNAi for Depletion of Enhancer RNAs in Primary Neurons.

Enhancer RNAs (eRNAs) are non-polyadenylated long noncoding RNAs (lncRNAs) transcribed from active enhancer regions and are increasingly recognized as key regulators of cell-type-specific gene expression. Noncoding RNAs exist in multiple forms and contribute to both transcriptional and posttranscriptional processes. Understanding their biological functions often requires selective depletion of individual RNA molecules. However, unlike protein-coding transcripts, eRNAs lack stable 3' ends, which complicates the use of conventional RNA interference (RNAi) approaches. These challenges are even more prominent in nondividing primary neurons, where transfection efficiency is inherently low. To address these limitations, this protocol outlines a lentiviral short hairpin RNAs (shRNAs)-based method for the efficient and specific depletion of eRNAs in primary cortical neurons. Step-by-step procedures are included for selecting shRNA target regions using nascent RNA sequencing data, generating shRNA-expressing lentiviruses, infecting primary neurons, and evaluating knockdown efficiency by RT-qPCR. This workflow offers a practical strategy for investigating eRNA function in postmitotic neurons and can be readily adapted to study enhancer-mediated transcriptional regulation in other nondividing cell types.