RNASeq of Porphyromonas gingivalis: Mouse skin abscess
mRNA from organisms is enriched using oligo(dT) beads. rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs(dUTP, dATP, dGTP and dCTP), RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Then followed by purification by AMPure XP beads, terminal repair, polyadenylation, sequencing adapter ligation, size selection and degradation of second-strand U-contained cDNA by the USER enzyme. The strand-specific cDNA library was generated after the final PCR enrichment.