1. mRNA Sample Preparation

For Cap type predictions in mRNA, the mRNA sample must first be prepared according to the following specifications:

1. Decapping: The m7G moiety at the 5' end of the mRNA must be removed through a decapping process.

2. Oligonucleotide Extension (OTE): A specific 52-oligonucleotide sequence must be ligated to the 5' end of each mRNA molecule. The OTE sequence is:

   5'-GCUUUCGUUCGUCUCCGGACUUAUCGCACCACCUAUCCAUCAUCAGUACUGU-3'
   

3. Sequencing: Samples should be sequenced using Oxford Nanopore Technologies (ONT) SQK-RNA004 chemistry.

By adhering to these preparation guidelines, researchers can maximize the accuracy and reliability of Capfinder's predictions, enabling deeper insights into RNA cap structures and their biological significance.