A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol.
| Title: | A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol. |
|---|---|
| Authors: | Anastasakis DG; Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892 MD, USA.; Jacob A; Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892 MD, USA.; Konstantinidou P; Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892 MD, USA.; Meguro K; Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892 MD, USA.; Claypool D; Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892 MD, USA.; Cekan P; MultiplexDX s.r.o., 841 04 Bratislava, Slovakia.; Haase AD; Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892 MD, USA.; Hafner M; Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892 MD, USA. |
| Source: | Nucleic acids research [Nucleic Acids Res] 2021 May 07; Vol. 49 (8), pp. e45. |
| Publication Type: | Journal Article; Research Support, N.I.H., Intramural |
| Language: | English |
| Journal Info: | Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE |
| Imprint Name(s): | Publication: 1992- : Oxford : Oxford University Press; Original Publication: London, Information Retrieval ltd. |
| MeSH Terms: | DNA, Complementary/*genetics ; Gene Expression Profiling/*methods ; High-Throughput Nucleotide Sequencing/*methods ; RNA-Binding Proteins/*metabolism ; Ribonucleoproteins/*metabolism; Cross-Linking Reagents/chemistry ; GTP Phosphohydrolases/chemistry ; Polymerase Chain Reaction/methods ; RNA/chemistry ; Ribonucleoproteins/genetics ; Thiouridine/chemistry ; Binding Sites ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Gene Library ; Humans ; Immunoprecipitation ; Oligonucleotides ; Protein Binding ; Sensitivity and Specificity ; Software ; Ultraviolet Rays |
| Abstract: | 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'end 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 10-100-fold.; (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.) |
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| Grant Information: | T32 GM140995 United States GM NIGMS NIH HHS |
| Substance Nomenclature: | 0 (Cross-Linking Reagents); 0 (DNA, Complementary); 0 (Oligonucleotides); 0 (RNA-Binding Proteins); 0 (Ribonucleoproteins); 13957-31-8 (Thiouridine); 63231-63-0 (RNA); EC 3.6.1.- (GTP Phosphohydrolases); EC 3.6.1.- (thioguanosine triphosphatase) |
| Entry Date(s): | Date Created: 20210127 Date Completed: 20210616 Latest Revision: 20221110 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC8096255 |
| DOI: | 10.1093/nar/gkab011 |
| PMID: | 33503264 |
| Database: | MEDLINE |
Journal Article; Research Support, N.I.H., Intramural