PRO-seq
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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:
- Detection of direct transcriptional effects of experimental perturbations
- Shorter treatment times with drug or protein degradation yield healthier cells which result in fewer confounding effects of cell stress in the data.
Applications
- Single nucleotide resolution of engaged and active RNAP across the genome
- Analysis of polymerase pausing and productive elongation
- Sensitive measurement of differential gene expression
- 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.
Sample Preparation
Required Input
| Method | Minimum # cells | Requested # of cells | Cell condition | Spike-in provided | Spike-in required |
|---|---|---|---|---|---|
PRO-seq | 1 million* | 1-3 million | frozen permeabilized cells | yes | yes |
* If cells are not limiting, we will use 1M permeabilized cells per sample for the nuclear run-on reaction but our protocol has performed robustly with 500k inputs using a wide variety of cell lines. Inputs lower than 500k may be possible but must be approved by us in advance. For low input samples, we may suggest a preliminary input titration experiment to determine the minimum number of cells needed.
Permeabilization Protocol
PRO-seq requires cells permeabilized using the protocol below. Preparing high quality cells is critical to obtain good results. The protocol below has worked on a wide variety of cell lines and tissue types and we can help troubleshoot issues with your sample preparation.
Cell Permeabilization protocol for PRO-seq
Tips
In addition to the protocol above, it may be helpful to consult our tips for preparing good quality samples.
Citation for NTC's PRO-seq method
Mimoso CA, Goldman SR.
PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.
Current Protocols 2023;3:e961. https://doi.org/10.1002/cpz1.961.
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Further Reading
Reimer KA, Mimoso CA, Adelman K, Neugebauer KM.
Co-transcriptional splicing regulates 3′ end cleavage during mammalian erythropoiesis.
Molecular Cell 2021. https://doi.org/10.1016/j.molcel.2020.12.018.
Kwak H, Fuda NJ, Core LJ, Lis JT.
Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing.
Science 2013;339:950–3. https://doi.org/10.1126/science.1229386.
Mahat DB, Kwak H, Booth GT, Jonkers IH, Danko CG, Patel RK, et al.
Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq).
Nature Protocols 2016;11:1455–76. https://doi.org/10.1038/nprot.2016.086.