Method Description
PRO-seq (Precision Run-On Sequencing) offers single nucleotide resolution of nascent RNA 3' ends. This information can be used to identify candidate enhancer RNAs and profile the immediate transcriptional effects of experimental perturbations such as drug-treatment, cell differentiation, or protein degradation.
The method involves permeabilization of cells followed by a nuclear run-on assay in which engaged RNA polymerase complexes incorporate a single biotinylated nucleotide into the nascent RNA chain. These labeled RNAs are enriched using streptavidin beads and sequencing of the final libraries reveals RNA 3’ ends at single nucleotide resolution.
The sensitivity of this method permits detection of changes in gene expression at timepoints earlier than possible with bulk RNA based methods like RNA-seq. This has two key benefits:
1. Detection of direct transcriptional effects of experimental perturbations
2. Shorter treatment times with drug or protein degradation yield healthier cells which result in fewer confounding effects of cell stress in the data.
Applications
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Single nucleotide resolution of engaged and active RNAP across the genome
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Analysis of polymerase pausing and productive elongation
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Sensitive measurement of differential gene expression
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Identification of enhancers
Example: PRO-seq applied to study PolI decay rates with triptolide treatment (from Elrod et al., 2019, Mol. Cell 76, 738–752 [2019])
Service Description
Users permeabilize and flash freeze cells according to the provided protocol (see: PRO-seq downloads). If your cells pass our quality control checks (sufficient number, >80% permeabilized, in single cell suspension, minimal debris), we will construct libraries, submit them for sequencing, analyze the data and return it to you. If cells fail QC, you will be notified and we will work with you to troubleshoot. The core provides appropriate spike-in cells for normalization purposes.
The preparation of high quality permeabilized cells is absolutely critical for obtaining good PRO-seq data. Our permeabilization protocol should be optimized for your specific cell type. Careful execution and practice before preparing your experimental samples will ensure good results and avoid delays caused by cells that do not pass QC.
See our tips for good experimental results.
PRO-seq library construction service includes:
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QC and spiking of user-provided permeabilized cells
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Nuclear run-on reaction
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PRO-seq library construction and QC
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High depth sequencing
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Return of fastq files of all sequencing data to user
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See Bioinformatics for data analysis options
User Provided Materials
2 million frozen, permeabilized cells (consult with us if this cell number is difficult to achieve)
Further Reading
Reimer KA, Mimoso CA, Adelman K, Neugebauer KM. Co-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis. Mol Cell. 2021 Mar 4;81(5):998-1012.e7. doi: 10.1016/j.molcel.2020.12.018. Epub 2021 Jan 12. PMID: 33440169.
Kwak H, Fuda NJ, Core LJ, Lis JT. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science. 2013 Feb 22;339(6122):950-3. doi: 10.1126/science.1229386. PMID: 23430654; PMCID: PMC3974810.
Mahat DB, Kwak H, Booth GT, Jonkers IH, Danko CG, Patel RK, Waters CT, Munson K, Core LJ, Lis JT. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc. 2016 Aug;11(8):1455-76. doi: 10.1038/nprot.2016.086. Epub 2016 Jul 21. PMID: 27442863; PMCID: PMC5502525.
