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Robust, Cost-Effective Profiling of RNA Binding Protein Targets With Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP)

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Robust, Cost-Effective Profiling of RNA Binding Protein Targets With Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP)

Eric L Van Nostrand et al. Methods Mol Biol.

Erratum in

Abstract

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.

Keywords: CLIP; CLIP-seq; RNA binding protein; RNA genomics; eCLIP; seCLIP; seCLIP-seq.

Figures

Figure 1
Figure 1. Schematic of seCLIP method
(a) Crosslinking of cultured cells (methods 3.0.1 - 3.0.2). (b) Lysis of crosslinked cells (methods 3.1.1). (c) RNA fragmentation with RNase (methods 3.1.2). (d) Immunoprecipitation of RBP-RNA complexes (methods 3.2.1 - 3.2.5). (e) Dephosphorylation of RNA fragments and ligation of 3′ RNA adapter (methods 3.4.1). (f) Polyacrylamide gel electrophoresis and membrane transfer (methods 3.5.1 - 3.5.5). (g) Mince preparative membrane into ~2mm squares (methods 3.5.6) (h) RNA isolation from membrane (methods 3.5.7 – 3.5.8). (i) Dephosphorylation of RNA fragments and ligation of 3′ RNA adapter for input samples (methods 3.6.1 – 3.7.3). (j) Reverse transcription of RNA (methods 3.8.1 – 3.8.2). (k) cDNA cleanup (removal of excess primers and RNA) (methods 3.8.3). (l) Ligation of 3′ DNA adapter (on-bead) and cleanup (methods 3.9.1 – 3.9.3). (m) PCR amplification of cDNA library and cleanup (Methods 3.10.2 – 3.11.2). (n) Final Structure of eCLIP library fragment. The unique molecular identifier or random-mer is shown in green and abbreviated as UMI.
Figure 2
Figure 2. Modified adaptor strategy for single-end enhanced CLIP (seCLIP)
(a) Schematic of adaptor sequences used in seCLIP. (b) Read density (shown as reads per million; RPM) observed in original, paired-end eCLIP (peCLIP) and seCLIP for RBFOX2 in HEK293T cells at EPB41L2 exon 13-14. Boxes below tracks indicate significantly enriched peaks after input normalization. (c) Required amplification observed using seCLIP and peCLIP adaptor strategies for two biological replicates profiling RBFOX2 in HEK293T. (d) Heatmap indicates correlation across experiments for read fold-enrichment in CLIP versus input, considering peaks identified in indicated experiments (y-axis). (e) Plot indicates enrichment for RBFOX2 binding motif UGCAUG at indicated positions around read start positions.
Figure 3
Figure 3. Genome of Acinetobacter johnsonii XBB1 in nitrocellulose membranes detected by RT—qPCR
(a) Sequencing reads from two eCLIP input libraries mapped to Acinetobacter johnsonii XBB1 (CP010350.1). (b) Bars indicate Cq from RT—qPCR performed using CP010350.1-specific qPCR primers on eCLIP RNA (methods 3.6.3) and supernatant samples (methods 3.3.1) from indicated cell types. Lower Cq reflects higher CP010350.1 signal. Numbers indicate replicate experiments. (c) Bars indicate RT—qPCR Cq for CP010350.1 from RNA isolated from nitrocellulose membranes via indicated method, PVDF membranes (RNA isolation by Trizol), paper (RNA isolation by Trizol), and H2O (Trizol extraction) (d) Bars indicate RT—qPCR Ct for CP010350.1 from RNA extracted from size-matched nitrocellulose membranes, in technical replicates. Symbols below indicate samples which were either RNase or DNase treated, and those with and without RT enzyme added (e) Bars indicate RT—qPCR Ct for CP010350.1 from RNA extracted from nitrocellulose membrane samples from five sources in technical replicates as follows: (A) commercial source A, (B) ThermoFisher iBlot (IB23001 lot 2NR26016-01), (C) GE Amersham Protran Premium (13600117 lot G6552142), (D) GE Amersham Protran Premium (1060008 lot G9931040), and (O) original commercial source. Error bars indicate standard deviation from RT—qPCR triplicate measurements.

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