title: Cannabis sativa Purple Kush genomic DNA library CS-PK_454_40kb-3
design: To construct the paired-end libraries for 454 sequencing, we followed the method described in the GS FLX Titanium 20 kb and 8 kb Span Paired End Library Preparation Method Manual (Roche, April 2009) with the following modifications. In Manual section 3.1, high-quality genomic DNA (45 µg) was fragmented using a Hydroshear (Digilab) with the large assembly and set to speed code 18 for 20 cycles. In section 3.3, the fragmented DNA was separated on a single lane of a 0.5% agarose gel (Megabase, Bio-Rad) in 1× TAE for 16 h at 14°C using a FIGE Mapper Electrophoresis System (Bio-Rad). Switch time ramp was set at 0.1-0.8 s with a linear shape and forward and reverse voltages were 180 V and 120 V, respectively. In section 3.4, four slices were cut from the one lane (13 kb, 20 kb, 30 kb and 40 kb). Libraries were multiplied at the DNA circularization (section 3.6) and library amplification (section 3.10) steps. For circularization of the 40 kb libraries, 600 ng were used instead of 300 ng (section 3.6.2). During the circularization incubation program (section 3.6.5), the 40 kb libraries were held at 37°C for 60 min instead of 45 min. To make the DNA beads, we followed the method described in the emPCR Method Manual – Lib-L LV GS FLX Titanium Series (Roche, October 2009 (Rev. Jan 2010)). For emPCR of paired-end libraries, the Live Amplification Mix (section 3.1.4) was modified and the heat denaturation (section 3.2.6) was omitted. To sequence, we followed the method described in the Sequencing Method Manual GS FLX Titanium Series (Roche, October 2009 (Rev. Jan 2010)) with software v2.5.3.