RNA-seq of M. bovis AF2122/97 rolling culture
mRNA from organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA isremoved 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 mRNAtemplate and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection andPCR enrichment.