Mapping Enhancer–Gene Interactions with Direct-Capture Targeted Perturb-seq (DC-TAP-seq) v1

Le résumé fourni par la source

Mapping enhancer-gene (E-G) connections is critical for dissecting the functional impact of noncoding variants in the human genome. Single-cell CRISPRi screens offer a powerful approach to identify these regulatory interactions, but existing methods such as Perturb-seq are limited by high sequencing costs and inefficient guide RNA capture across diverse cell types. Here, we decribe a detailed protocol for Direct-Capture Targeted Perturb-seq (DC-TAP-seq) — a cost-effective and scalable approach that integrates TAP-seq1 with direct capture of Pol III-derived guide RNAs2 to sensitively screen thousands of E-G connections across the genome. This protocols describes pooled cloning of gRNAs into an sgOpti-CS vector, high MOI lentiviral transduction, cell preparation for single-cell RNA-seq, and targeted amplification of both transcripts and guide RNAs. DC-TAP-seq enables large-scale mapping of E-G interactions with reduced cost and improved sensitivity, and is broadly applicable for studying E-G regulation in diverse human cell types.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.