{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T05:01:55Z","timestamp":1791435715635,"version":"4.3.3"},"reference-count":38,"publisher":"Oxford University Press (OUP)","issue":"8","funder":[{"DOI":"10.13039\/100000069","name":"National Institute of Arthritis and Musculoskeletal and Skin Diseases","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000069","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000062","name":"National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,5,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3\u2032end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10\u2013100-fold.<\/jats:p>","DOI":"10.1093\/nar\/gkab011","type":"journal-article","created":{"date-parts":[[2021,1,6]],"date-time":"2021-01-06T22:27:05Z","timestamp":1609972025000},"page":"e45-e45","source":"Crossref","is-referenced-by-count":53,"title":["A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol"],"prefix":"10.1093","volume":"49","author":[{"given":"Dimitrios G","family":"Anastasakis","sequence":"first","affiliation":[{"name":"Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexis","family":"Jacob","sequence":"additional","affiliation":[{"name":"Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parthena","family":"Konstantinidou","sequence":"additional","affiliation":[{"name":"Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuyuki","family":"Meguro","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Duncan","family":"Claypool","sequence":"additional","affiliation":[{"name":"Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pavol","family":"Cekan","sequence":"additional","affiliation":[{"name":"MultiplexDX s.r.o., 841 04 Bratislava,\u00a0Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1120-9013","authenticated-orcid":false,"given":"Astrid\u00a0D","family":"Haase","sequence":"additional","affiliation":[{"name":"Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4336-6518","authenticated-orcid":false,"given":"Markus","family":"Hafner","sequence":"additional","affiliation":[{"name":"Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892\u00a0MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2021,1,27]]},"reference":[{"key":"2021050500283769700_B1","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1146\/annurev-biochem-080111-092106","article-title":"The clothes make the mRNA: past and present trends in mRNP fashion","volume":"84","author":"Singh","year":"2015","journal-title":"Annu. 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