Magbead binding kit

The purified SMRTbell templates were bound to v2 sequencing primers and polymerases by using the DNA/Polymerase Binding Kit P6 (P/N 100-356-300). The conditions for annealing of the primers and binding of polymerase were determined using the PacBio's Binding Calculator in RS Remote. The polymerase-template complexes were bound to magbeads using the PacBio MagBead Binding Kit. The samples were analyzed on an Agilent 2100 bioanalyzer. Sequencing was carried out by using the PacBio RS II sequencer with DNA Sequencing Reagent 4.0 (P/N 100-356-200). The length of each movie was 360 min. We applied the PacBio Iso-Seq protocol (SMRT Analysis version v2.3.0.; Chaisson and Tesler, 2012 (link)) for the analysis of the HSV-1 transcriptome.

Target amplicons of equal size were pooled equimolar and a SMRT bell library was prepared as recommended by Pacific Biosciences (10 kb Template Preparation and Sequencing with Low-Input DNA) without an initial fragmentation. Raw sequence data is available upon request. Libraries were quantified by fluorometry and quality was assessed by capillary electrophoresis (Agilent DNA 12000 reagents and chips, Agilent). SMRT bell templates were bound to polymerases using the DNA/polymerase binding kit P6 and v2 primers. Polymerase-template complexes were bound to magnetic beads with the Magbead Binding Kit and sequencing was done on the PacBio RS II sequencer with C4 sequencing reagents with a movie length of 180 or 360 min. Cluster analysis to extract haplotypes of individuals was done with the in house algorithm (Wenzel and Pannes unpublished). Amplicons were split according to the 16mer barcodes of PacBio. Additionally amplicons were split with a Perl script.

The Poly(A⁺) fractions of total RNAs were quantified through use of the Qubit RNA HS Assay Kit (Life Technologies), followed by conversion to cDNAs with the SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies; the included first strand enzyme was changed to SuperScript III Reverse Transcriptase). The reverse transcription (RT) reactions were primed with Anchored Oligo(dT)₂₀ primers (Life Technologies). The cDNAs obtained were quantified with the Qubit HS dsDNA Assay Kit (Life Technologies).
SMRTbell sequencing libraries were generated by using the PacBio DNA Template Prep Kit 2.0 and the Pacific Biosciences template preparation and sequencing protocol for Very Low (10 ng) Input 2 kb libraries with carrier DNA (pBR322, Thermo Scientific). SMRTbell templates were bound to polymerases by using the DNA polymerase binding kit XL 1.0 (part #100-150-800) and v2 primers. The polymerase-template complexes were bound to magbeads with the Pacific Biosciences MagBead Binding Kit. The SMRTBell libraries were analyzed for length and concentration through use of the Agilent 2100 Bioanalyzer. DNA sequencing was carried out with a Pacific Biosciences RS II sequencer using P5-C3 chemistry. Movie lengths were 180 min.