5′-GRO-Seq

5′ Global Run-on Sequencing

5′-GRO-Seq maps the sequences of nascent RNA with a 7-methylguanylate (m7G) cap at any given time using labeled nucleotides (Lam et al., 2013). This method was originally developed to map and detect instabilities in TSS due to the presence of enhancer RNA. Much like GRO-seq, 5′-GRO-Seq starts with the addition of Br-UTP and sarkosyl to the lysed nuclear material. The Br-UTP acts as a marker for isolating the RNA, while sarkosyl inhibits binding of additional RNAPII to the DNA. After the reaction is stopped, DNase I is added and the RNA products fragmented. The 3′ ends of RNA are dephosphorylated with T4 PNK and fragments containing bromouridine are captured with anti-BrdU antibodies. The isolated RNAs are dephosphorylated with CIP, and the 5′ caps are removed using TAP. Next, 3′ and 5′ adapters are ligated using RNA ligase 2 and RNA ligase, respectively. The fragments are reverse-transcribed, the resultant cDNA is isolated and amplified, and the cDNA is size-selected for 60_110 bp fragments. The fragments are isolated from the gel and sequenced.

Advantages:

• Maps nascent capped 5′ RNA sequence at any given time
• Determines activity of transcription sites
• No prior knowledge of transcription sites needed

Disadvantages:

• Limited to cell cultures and other artificial systems, due to the requirement for incubation in the presence of labeled nucleotides

Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Lam M. T., Li W., Rosenfeld M. G. and Glass C. K. Enhancer RNAs and regulated transcriptional programs. Trends Biochem Sci. 2014;39:170-182



References:

Duttke S. H., Lacadie S. A., Ibrahim M. M., et al. Human promoters are intrinsically directional. Mol Cell. 2015;57:674-684

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History: 5′-GRO-Seq

Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide