Bison_x_Simmental F1 Oxford Nanopore Ultra-long
Template for long read sequencing on the GridION instrument was prepared using the Ligation Sequencing Kit LSK-109 (Oxford Nanopore Technologies, Oxford, U.K.).  Briefly, 10 ug of DNA was sheared by passing 5 times through a 26 gauge needle and concentrated by addition of 1 volume of AMPureXP bead solution.  After collecting the beads by magnet, the beads were washed 2 times with 0.5 mls 80% ethanol and dried for 2 minutes.  The beads were suspended in 51 uL EB and DNA eluted at 37C for 5 minutes.  The sheared DNA was transferred to a tube with wide-bore pipette tips and mixed with a PEG/NaCl precipitation buffer (9% PEG8000, 1M NaCl and 10 mM Tris-Cl ph 8.0). The mixture was incubated at room temperature for 30 minutes and was centrifuged at 13.5 k RPM for 30 minutes. The supernatant was subjected to two, 200 ul, 70% ethanol precipitations with centrifugation, and the pellet was eluted in 41 ul of 10 mM Tris-Cl ph 8.0. All other methods in the LSK109 library preparation protocol were followed, apart from an increase in the room temperature incubation of the ligation reaction to 60 minutes at room temperature. Sample was sequenced on a GridION instrument using R9.4.1 flow cells.