5'-P TT DBE+ rep 2
For 5'-P library preparation, unfragmented total RNA samples from cells or purified U2 U5 U6 NTC complex were treated (DBE+) or not (DBE-) with recombinant DBE and then used directly for library construction employing a protocol similar to that described for making degradome libraries (Moran et al., 2014), except that 5' adapter (aka, RNA linker) ligations were carried out for 2 hours at 25 C and ethanol precipitations were substituted for cleanup steps calling for Agencourt RNAClean XP beads. Also the RNA linker was modified to contain a region of nine random nucleotides in order to label each captured RNA fragment with a unique molecular identifier (UMI). RT and PCR primer sequences were exactly as in (Moran et al., 2014).