5'-GRO-Seq
5'-GRO-Seq
5' global run-on sequencing (5'-GRO-Seq) maps the sequences of nascent RNA products with 5'7-methyl-guanylated caps at any given time using labelled nucleotides. This method was originally developed to map and detect instabilities in transcription start sites (TSS) due to the presence of enhancer RNA. Much like global run-on sequencing (GRO-Seq), 5'-GRO-Seq starts with the addition of bromo-uridinetriphoshate (Br-UTP) and sarkosyl to the lysed nucleic material. The Br-UTP acts as a marker for isolating the RNA while sarkosyl inhibits binding of additional RNA polymerase II to the DNA. After the reaction is stopped, DNase is added and the RNA products fragmented. 3'-ends of RNA are dephosphorylated with polynucleotide kinase and fragments containing Br-UTP captured with anti-BrdUTP antibodies. Isolated RNAs are dephosphorylated with calf intestinal-phosphatase (CIP) and fragments with 5' caps de-capped using tobacco acid pyrophosphatase (TAP). 3' and 5' adaptors are ligated using RNA ligase 2 and RNA ligase respectively. The fragments are reverse transcribed, resultant cDNA isolated and amplified, and size selected for 60-110 bp sized fragments. The fragments are isolated from the gel and sequenced.
Pros:
- Maps nascent capped 5' RNA sequence at any given time
- Determines activity of transcription sites
- No prior knowledge of transcription sites needed
Cons:
- Limited to cell cultures and other artificial systems due to incubation with labelled nucleotides