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. 2019 Jan 31;47(6):e33. doi: 10.1093/nar/gkz049

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — A highly efficient covalent linkage-based SpyCLIP platform for the characterization of protein–RNA interactions. (A) Schematic representation of the SpyCLIP procedure. (B) Schematic representation of the Spy-tagged RBP structure and mechanism of the SpyTag-SpyCatcher covalent interaction. (C) Cellular localization of Spy-tagged RBPs ectopically expressed in Lenti-X 293T cells. (D) The modified SpyCatcher protein efficiently formed a stable complex with Spy-tagged PTBP1 in SpyCLIP lysis buffer. (E) The covalent SpyTag-SpyCatcher pull-down system can withstand harsh washing conditions, including buffers containing 8 M urea, 1% SDS, 2 M NaCl or 6 M guanidine hydrochloride. (F) Comparison of expression levels of Dox-induced Spy-tagged PTBP1 with its endogenous counterpart in Lenti-X 293T cells, and 1 ng/ml Dox was used for all RBPs used in this study. The PTBP1 band in the No Dox lane is due to leak expression of the tet-on system. (G) Amplification of PTBP1 SpyCLIP libraries using different PCR cycles. Twenty PCR cycles can provide a sufficient amount of cDNA for deep sequencing in a relatively small volume and did not increase the percentage of PCR duplicates; hence, 20 PCR cycles were chosen for library preparations. The region marked by the rectangle (160–180 bp) was recovered for deep sequencing.