Nuclear run-on sequencing

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Global run-on sequencing (GRO-seq) is a special method for RNA sequencing in vitro , in which RNA segments with transcriptionally active RNA polymerase II are identified in the genome . The method is based on the nuclear run-on assay (NRO). The advantage over the methods based on chromatin immunoprecipitation is that only actually transcribing areas of the RNA are specifically detected. This enables a “snapshot” of the current genome-wide transcription.

The goal of an NGO run is to isolate RNA that is being produced at a given moment. For this purpose, several million cell nuclei are isolated, existing (endogenous) free nucleotides are removed and then labeled nucleotides (e.g. with 5-bromouracil ) are added. Short transit times ensure that only regions in the direction of transcription (towards the 3 'end of the DNA strand, referred to as downstream in laboratory jargon ) of the bound RNA polymerase II are transcribed. The anionic detergent Sarkosyl is often added in order to prevent renewed initiation of the RNA polymerase II. Isolated NRO-RNA is fragmented and purified with magnetic beads coated with anti-BrU antibodies (small magnetic particles which bind to the molecule via the antibodies and which then enable separation by means of a magnetic field). The RNA is then prepared for sequencing and then sequenced.

The advantages of the GRO-seq include the differentiation between transcription on the (+) and (-) strand and the high-resolution, global recording of the relative activity of RNA polymerase II. The disadvantage is that it is carried out in vitro , a potential new initiation during the run- on -steps and artifacts that can arise during the preparation of the cell nuclei.

Due to the high resolution of sequencing methods, short-lived and only small numbers of RNAs such as. B. non-coding enhancer RNA (eRNA) can be identified.

Bibliography

  1. ↑ a b Leighton J. Core, Joshua J. Waterfall, John T. Lis: Nascent RNA sequencing Reveals widespread pausing and divergent initiation at human promoter , Science 322 pp 1845 to 1848 (2008) doi : 10.1126 / science.1162228
  2. ↑ cf. Stephen T. Smale: Nuclear Run-On Assay. Cold Spring Harbor Protocols 2009. doi : 10.1101 / pdb.prot5329
  3. ↑ Wang et al. , Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA , Nature 474 pp. 390-394 (2011)