Abstract
Many features of how gene transcription occurs in human cells remain unclear, mainly because of a lack of quantitative approaches to follow genome transcription with nucleotide precision in vivo. Here we present a robust genome-wide approach for studying RNA polymerase II (Pol II)–mediated transcription in human cells at single-nucleotide resolution by native elongating transcript sequencing (NET-seq). Elongating RNA polymerase and the associated nascent RNA are prepared by cell fractionation, avoiding immunoprecipitation or RNA labeling. The 3′ ends of nascent RNAs are captured through barcode linker ligation and converted into a DNA sequencing library. The identity and abundance of the 3′ ends are determined by high-throughput sequencing, which reveals the exact genomic locations of Pol II. Human NET-seq can be applied to the study of the full spectrum of Pol II transcriptional activities, including the production of unstable RNAs and transcriptional pausing. By using the protocol described here, a NET-seq library can be obtained from human cells in 5 d.
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Acknowledgements
We thank J. di Iulio and S. Maleri for help developing this approach. We thank J. di Iulio, H. Landry and A. Snavely for critical comments on the manuscript. This work was supported by US National Institutes of Health National Human Genome Research Institute (NHGRI) grant R01HG007173 to L.S.C.; a Damon Runyon Dale F. Frey Award for Breakthrough Scientists (to L.S.C.); and a Burroughs Wellcome Fund Career Award at the Scientific Interface (to L.S.C.). A.M. was supported by Long-Term Postdoctoral Fellowships of the Human Frontier Science Program (HFSP) (LT000314/2013-L) and the European Molecular Biology Organization (EMBO) (ALTF858-2012).
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A.M. performed experiments and prepared figures. A.M. and L.S.C. wrote the manuscript.
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Supplementary Figure 1 Representative gel to monitor the efficiency of DNA linker ligations.
The RNA control oligo oGAB11 (lane 2) was ligated to the DNA linker (no barcode, lane 3) and to the Barcode DNA linker (lane 4). The ligation was performed as described in Procedure steps 25 to 28. The samples were separated on a 15% (wt/vol) polyacrylamide TBE-Urea gel as described in Procedure steps 38 to 43 of this protocol. The ligation efficiency is >95% for both ligations (lanes 3 and 4) as determined by image quantification using ImageJ 1.47v software.
Supplementary Figure 2 Full-size blots of the western analysis shown in Fig. 2.
For more details please see legend of Fig. 2.
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Mayer, A., Churchman, L. Genome-wide profiling of RNA polymerase transcription at nucleotide resolution in human cells with native elongating transcript sequencing. Nat Protoc 11, 813–833 (2016). https://doi.org/10.1038/nprot.2016.047
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DOI: https://doi.org/10.1038/nprot.2016.047
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