Hifi taq polymerase

To identify novel human fusion transcripts, specific primers were designed to match the 5’ and 3’ ends of a given fusion transcript, using a primer-designing software [53 ]. 5 μl of the cDNAs generated above were used as template for end-point PCR, which was carried out using HiFi Taq polymerase (Invitrogen, Carlsbad, CA, USA) for 35 cycles of 94°C for 15”, 60-68°C for 15” and 68°C for 2-5 min. The PCR products of KANSARL isoforms 1 and 2 were separated on 2% agarose gels, recovered and cloned into TOPO pCR2.1 vector as suggested by manufacturer. After transformation and incubation at 37oC overnight, the plasmids with inserts were isolated and sequenced. The sequences were then verified by blast and manual inspection. For genomic fusion validation, genomic DNA from HeLa-3 cells was used as template for end-point PCR using the Phusion PCR kit (NEB), with an annealing temperature at 60°C.

To ensure culture purity before whole-genome sequencing, rpoB sequencing and 16S rRNA analysis were performed. Briefly, genomic DNA was isolated from 5 ml TYES cultures of the parent and passaged strains of F. psychrophilum with QIAamp DNA Mini Kit (QIAgen) according to the manufacturer’s instructions. External primers for rpoB amplification were 5′-AAAATCGGAACGGATTACGG-3′ and 5′-TTTTGAATTGTTTTTAAAGAGGTATTG-3′, and PCR involved 35 cycles of 94 °C for 30 s, 45 °C for 30 s and 68 °C for 4 min. Primers used to amplify internal rpoB segments for sequencing were described previously by Gliniewicz and coworkers [14 (link)]. All reactions included 2 mM MgSO₄, 1 × PCR buffer, 0.2 mM dNTP mixture, 1 U of HiFi Taq polymerase (Invitrogen) with 5 ng DNA template and 0.1 μM of each primer in 50 μl reaction volume. 16S rRNA analysis was performed according to method described by Soule and coworkers with 16S_336fwd and 16S_517rvs primers used for target amplification and MaeIII digestion of amplified 16S rRNA to distinguish CSF 259–93 and THC 02–90 strains [19 (link)].