Genome-wide Mapping of Uncapped and Cleaved Transcripts
GMUCT is a method for constructing sequencing libraries made up of decapped or cleaved mRNAs (Gregory et al., 2008). 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 (Willmann et al., 2014).
GMUCT 1.0: Starting with total RNA, mRNA is isolated by poly(A) selection. Next, 5′ RNA adapters are ligated and the RNA is reverse-transcribed. The resultant first strand of cDNA is amplified using oligo(dT) and 5′-adapter primers. The double-stranded cDNA is fragmented, ligated to 3′- and 5′-sequencing adapters, PCR-amplified, and sequenced.
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.
- Sequences RNA degradation intermediates and uncapped RNAs
- GMUCT 2.0 only takes 2-3 days and requires just 5 µg of total RNA
- Can be modified to study cleavage of miRNA or siRNA targets
- Minimum size of 135 bp
Illumina Library prep and Array Kit Selector
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
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