Global Run-On Sequencing (GRO-Seq)

Global Run-On Sequencing (GRO-Seq), also known as Nascent RNA Sequencing Analysis (NRSA), is a next-generation sequencing (NGS)-based method to detect transcripts that are initiated at the time of nuclei isolation (commonly referred to as nascent RNA). In a GRO-Seq protocol, nascent RNA is isolated and can be ultimately converted into a DNA library suitable for deep sequencing. We provide comprehensive GRO-Seq services to help you identify nascent transcription changes for genes and enhancers.

Overview

Gro-seq is a specialized method for measuring nascent RNA that can be used for systematic identification of eRNA molecules. The method involves rapid freezing of targeted cells to ensure the congelation of the transcription complex in the nucleus, followed by the extraction of the nucleus. The Run-On buffer is then added to restore RNA transcription. And with the presence of anti-BrdU antibody, BrU tagged nascent RNA is extracted and ready for high-throughput sequencing. The alteration of nascent RNA directly reflects multiple mechanisms of transcriptional regulation. With the help of GRO-Seq, the ongoing transcription can be observed at the genomic level, which is a high-resolution mapping of the location, number, and orientation of all participating RNA polymerases. Thus, GRO-Seq can be used to detect direct target genes and novel transcripts (ncRNA included).

Features

High Throughput High Accuracy High Repeatability One-stop Service
Obtain more than 10 million sequence information in one sequencing. Accurately detect gene expression in real-time;
Precisely detect the transcription location and direction.
Deep sequencing ensures the randomness of detection and does not require technical repetition. Provides one-stop services for library construction, sequencing, sample QC and data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

Size selection; strand-specific cDNA libraries.

Sequencing

3. Sequencing

Illumina Next Seq; SE 75.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Bioinformatics Analysis Pipeline

Global Run-On Sequencing (GRO-Seq)

In-depth data analysis:

  • Data quality control;
  • Reference-based mapping;
  • Meta-analysis near the transcription start sites (TSSs);
  • Transcriptional pausing analysis;
  • eRNA analysis;
  • Differential gene analysis;
  • GO and KEGG pathway analysis.

Sample Requirements

Minimum number of cells: 1×107

Sample storage: The nucleus is recommended to be separated and frozen in liquid nitrogen and transported in dry ice. Otherwise, living cells (with culture medium) shall be submitted and transported at room temperature. If pre-treatment is required, please provide detailed description and related materials.

Shipping Method: When shipping nucleus samples, the shipment is recommended to contain 5-10 pounds of dry ice per 24 hours. When shipping living cell samples, the samples can be transported at room temperature.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

Reference:

  1. Wang J, Zhao Y, Zhou X; et al. Nascent RNA sequencing analysis provides insights into enhancer-mediated gene regulation. BMC Genomics. 2018, 19(1):633.
* For Research Use Only. Not for use in diagnostic procedures.


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