GMUCT 2.0

GMUCT 2.0

GMUCT is a method for constructing sequencing libraries made up of decapped or cleaved mRNAs. There are 2 versions of GMUCT: version 1.0 was developed in 2008, while a more streamlined and efficient version 2.0 was designed in 2013. GMUCT 2.0 significantly decreases library preparation time by 3 days (it takes 2–3 days instead of 5–6 days) and requires 10 times less starting total RNA (5 µg instead of 50 µg.

GMUCT 2.0: This version starts out similar to GMUCT 1.0, but deviates after the ligation of 5' RNA adapters to poly(A) RNAs. Another round of poly(A) selection is performed to further purify the desired mRNA away from any unligated RNA. RT is performed using primers with 3' adapters on their 5' terminus and a random hexamer on the 3' end. This modification adds 3' adapters during RT, resulting in cDNA strands flanked with adapters at both ends. The cDNA is PCR-amplified, to add sequencing indexes, and sequenced.

Pros:

• Sequences RNA degradation intermediates and uncapped RNAs
• GMUCT 2.0 only takes 2-3 days and 5 µg of total RNA
• Can be modified to study cleavage of miRNA or siRNA targets

Cons:

• Minimum size of 135 bp

• GMUCT 2.0: Willmann M. R., Berkowitz N. D. and Gregory B. D. (2014) Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes--GMUCT 2.0. Methods 67: 64-73

1. Ma X., Tang Z., Qin J. and Meng Y. The use of high-throughput sequencing methods for plant microRNA research. RNA Biol. 2015;12:709-719

1. Vandivier L. E., Campos R., Kuksa P. P., Silverman I. M., Wang L. S. and Gregory B. D. Chemical Modifications Mark Alternatively Spliced and Uncapped Messenger RNAs in Arabidopsis. Plant Cell. 2015;27:3024-3037