Genome-wide analysis of polyadenylation events in Schmidtea mediterranea
This protocol was original developed by Jan et al., 2010. The protocol involves isolation of total RNA followed by ligation of splint oligos bound to biotin and base pairs with Poly A tail.  The splint oligo ligated mRNA was partially digested with RNase T1, which cleaves at the G residue. The partially digested mRNA was bound to the streptavidin beads via biotin followed by primer annealing and reverse transcription using dTTP nucleotide, which fill-in the poly A stretch. The reverse transcribed products were digested with RNAseH, which get rid of DNA:RNA hybrid leaving single stranded RNA with a small stretch of A residue at the 5’ end. Later 3’ and 5’ adaptor were ligated to the single stranded RNA followed by RT-PCR and deep sequencing using illumina genomic DNA primers on Illumina, GAIIx platform.
inStem, NCBS
3P sexual R2