title: PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites.
abstract: The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale. PAR-CLIP involves irreversible UV-mediated crosslinking of RNAs labeled with photoreactive nucleosides to interacting proteins, followed by stringent purification steps and the conversion of crosslinked RNA into small RNA cDNA libraries compatible with next-generation sequencing. The defining hallmark of PAR-CLIP is a diagnostic mutation at the crosslinking site that is introduced into cDNA during the library preparation process. This feature allows for efficient computational removal of contaminating sequences derived from non-crosslinked fragments of abundant cellular RNAs. In the following, we present two different step-by-step procedures for PAR-CLIP, which differ in the small RNA cDNA library preparation procedure: (1) Standard library preparation involving gel size selections after each enzymatic manipulation, and (2) A modified PAR-CLIP procedure ("on-beads" PAR-CLIP), where most RNA manipulations including the necessary adapter ligation steps are performed on the immobilized RNP. This streamlined procedure reduces the protocol preparation time by three days compared to the standard workflow.
authors.affiliation: Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, 50 South Drive, Bethesda, MD 20892, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Benhalevy
authors.affiliation: Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, 50 South Drive, Bethesda, MD 20892, USA.
authors.forename: Hannah L
authors.initials: HL
authors.lastname: McFarland
authors.affiliation: Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, 50 South Drive, Bethesda, MD 20892, USA.
authors.forename: Aishe A
authors.initials: AA
authors.lastname: Sarshad
authors.affiliation: Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, 50 South Drive, Bethesda, MD 20892, USA. Electronic address: markus.hafner@nih.gov.
authors.forename: Markus
authors.initials: M
authors.lastname: Hafner
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D009154:Mutation; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software; D013891:Thiouridine / Q000737:chemistry / Q000378:metabolism*; D059467:Transcriptome; D014466:Ultraviolet Rays; D051840:Y-Box-Binding Protein 1 / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D010761:Phosphorus Radioisotopes; D016601:RNA-Binding Proteins; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D013891:Thiouridine; D012313:RNA; D012260:Ribonucleases
keywords: Next-generation sequencing; PAR-CLIP; Posttranscriptional Gene Regulation; RNA binding protein; Small RNA cDNA library