IsoSeq of Histophilus somni
PolyA-tailing of approximately 10 g rRNA-depleted bacterial RNA was performed using a kit (PAP5104H) as recommended by the manufacturer (Lucigen, Middleton WI USA).  Tailed RNA was purified using RNeasy MinElute Cleanup Kit as recommended by the manufacturer (Qiagen, Hilden Germany). Conversion of the tailed RNA to cDNA was performed using the SMARTer kit and tailed polyT primer as recommended by the manufacturer (Clontech, Mountain View CA USA). Test amplifications of cDNA were performed to determine optimal number of cycles for amplification retaining the input RNA size distribution.  The cDNA was then amplified for 12 cycles using the primers in the SMARTer kit, size selected on an ELF instrument (Sage Science, Beverly MA USA) to target fragments in the 1-2 kb range and fractions falling within that range were pooled and another round of 12 cycles of amplification performed.  The amplified cDNA was converted into SMRTbell libraries using the SMRTbell Template Prep Kit 1.0 as directed by the manufacturer (Pacific BioSciences, Menlo Park CA USA) and sequenced on an RSII instrument using P6 chemistry.