TY - JOUR
T1 - Proximity-CLIP and Expedited Non-Radioactive Library Preparation of Small RNA Footprints for Next-Generation Sequencing
AU - Anastasakis, Dimitrios
AU - Benhalevy, Daniel
AU - Hafner, Markus
N1 - Publisher Copyright:
Published 2020. This article is a US Government work and is in the public domain in the USA.
PY - 2020/6/1
Y1 - 2020/6/1
N2 - During the course of their life cycle, most RNAs move between several cellular environments where they associate with different RNA binding proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity-CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA crosslinking to simultaneously profile the proteome, including RBPs and the RBP-bound transcriptome, in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity-CLIP along with a simplified non-radioactive, small-RNA cDNA library preparation protocol. Published 2020 U.S. Government. Basic Protocol 1: Cell culture, 4SU labeling, proximity biotinylation, and crosslinking. Basic Protocol 2: Cell extraction, streptavidin affinity purification, and on-beads trypsinization. Basic Protocol 3: RNA footprints cDNA library preparation. Support Protocol: Preparation of RNA-seq libraries from intact RNA.
AB - During the course of their life cycle, most RNAs move between several cellular environments where they associate with different RNA binding proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity-CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA crosslinking to simultaneously profile the proteome, including RBPs and the RBP-bound transcriptome, in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity-CLIP along with a simplified non-radioactive, small-RNA cDNA library preparation protocol. Published 2020 U.S. Government. Basic Protocol 1: Cell culture, 4SU labeling, proximity biotinylation, and crosslinking. Basic Protocol 2: Cell extraction, streptavidin affinity purification, and on-beads trypsinization. Basic Protocol 3: RNA footprints cDNA library preparation. Support Protocol: Preparation of RNA-seq libraries from intact RNA.
KW - PAR-CLIP
KW - Proximity-CLIP
KW - RNA localization
KW - RNA processing intermediates
KW - RNA regulatory elements
KW - RNA-protein interactions
KW - non-radioactive small RNA cDNA library preparation
KW - subcellular RNA biology
UR - http://www.scopus.com/inward/record.url?scp=85085157742&partnerID=8YFLogxK
U2 - 10.1002/cpmb.120
DO - 10.1002/cpmb.120
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C2 - 32438484
AN - SCOPUS:85085157742
SN - 1934-3639
VL - 131
JO - Current Protocols in Molecular Biology
JF - Current Protocols in Molecular Biology
IS - 1
M1 - e120
ER -