{"doi":"10.1371/journal.pone.0315595","title":"Differential impact of divalent metals on native elongating transcript sequencing (NET-seq) protocols for RNA polymerases I and II","abstract":"Throughout all domains of life, RNA polymerases (Pols) synthesize RNA from DNA templates, a process called transcription. During transcription, Pols require divalent metal cations for nucleotide addition and cleavage of the nascent RNA after misincorporation or polymerase stalling. Recently, several next-generation sequencing techniques have emerged to study transcription at single-nucleotide resolution in vivo. One such technique, native elongating transcript sequencing (NET-seq), allows for isolation of transcription elongation complexes associated with a specific Pol, defining polymerase occupancy on the DNA template. Originally developed to study RNA polymerase II (Pol II), NET-seq has been adapted for RNA polymerase I (Pol I) and bacterial RNA polymerase. We recently optimized Pol I NET-seq in Saccharomyces cerevisiae, however, we omitted nucleases and their metal cofactors, which are commonly used in Pol II NET-seq. Here, we investigated the effect of CaCl2 ± MNase and MnCl2 ± DNase I on Pol I occupancy. We found that exposure of Pol I to CaCl2 and MnCl2 during NET-seq caused a significant reduction in immunoprecipitation of nascent rRNA compared to the untreated control samples, with a more severe effect when incubated with MnCl2 vs. CaCl2. Surprisingly, in contrast to the Pol I results, we found that metal treatment during Pol II NET-seq did not have a significant effect on nascent transcript capture. Taken together, these observations reinforce the conclusion that transcription elongation complexes formed by Pols I and II have unique characteristics and emphasize the need to carefully consider experimental conditions deployed in all stages of nucleic acid library generation.","journal":"PLoS ONE","year":2025,"id":562019,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9514,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":504476,"name":"David A. Schneider","orcid":"0000-0003-0635-5091","position":1,"is_corresponding":false},{"id":650677,"name":"Abigail K. Huffines","orcid":null,"position":0,"is_corresponding":true}],"reference_count":38,"raw_metadata":null,"created_at":"2026-07-19T02:56:01.883848Z","pmid":"39946358","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}