Maize Genomic DNA Preparation for SMRT Sequencing

DNA samples for SMRT sequencing were prepared using maize inbred line B73 from NCRPIS (PI550473), grown at University of Missouri. Seeds of this line were deposited at NCRPIS (tracking number PI677128). Etiolated seedlings were grown for 4–6 days in Pro-Mix at 37 °C in darkness to minimize chloroplast DNA. Batches of ~10 g were snap-frozen in liquid nitrogen. DNA was extracted following the PacBio protocol ‘Preparing Arabidopsis Genomic DNA for Size-Selected ~20 kb SMRTbell Libraries’ (http://www.pacb.com/wp-content/uploads/2015/09/Shared-Protocol-Preparing-Arabidopsis-DNA-for-20-kb-SMRTbell-Libraries.pdf).
Genomic DNA was sheared to a size range of 15–40 kb using either G-tubes (Covaris) or a Megarupter device (Diagenode), and enzymatically repaired and converted into SMRTbell template libraries as recommended by Pacific Biosciences. In brief, hairpin adapters were ligated, after which the remaining damaged DNA fragments and those without adapters at both ends were eliminated by digestion with exonucleases. The resulting SMRTbell templates were size-selected by Blue Pippin electrophoresis (Sage Sciences) and templates ranging from 15 to 50 kb, were sequenced on a PacBio RS II instrument using P6-C4 sequencing chemistry. To acquire long reads, all data were collected as either 5- or 6-h sequencing videos.