Rbk004 kit

Samples are library prepped depending on whether an R9 or R10 flowcell is to be used. For R9 flowcells, the Oxford Nanopore RBK004 kit was used, using 600 ng input material and following manufacturer’s instructions for other steps. For R10 flowcells, we used the Oxford Nanopore technologies RBK114-24 kit, but with an adjusted protocol: for optimal results (size distribution centred around 5 kb) 3,500 ng of input material in 50 μl is first sheared using a G-Tube (Covaris SKU: 520079), centrifuging at 7,200 RPM (6,000g) in a fixed rotor tabletop centrifuge. Subsequently 7.5 μl of the sheared input material is used for tagmentation with 2.5 μl indexing mix. Alternatively, we obtain similar results (but with wider size distributions) using 200 ng input material for the tagmentation without an added fragmentation step. For both protocols, we omit the AMPure purification, and after tagmentation directly proceed with adapter ligation.

Samples are library prepped depending on whether an R9 or R10 flowcell is to be used. For R9 flowcells, the Oxford Nanopore RBK004 kit was used, using 600 ng input material and following manufacturer’s instructions for other steps. For R10 flowcells, we used the Oxford Nanopore technologies RBK114-24 kit, but with an adjusted protocol: for optimal results (size distribution centred around 5 kb) 3,500 ng of input material in 50 μl is first sheared using a G-Tube (Covaris SKU: 520079), centrifuging at 7,200 RPM (6,000g) in a fixed rotor tabletop centrifuge. Subsequently 7.5 μl of the sheared input material is used for tagmentation with 2.5 μl indexing mix. Alternatively, we obtain similar results (but with wider size distributions) using 200 ng input material for the tagmentation without an added fragmentation step. For both protocols, we omit the AMPure purification, and after tagmentation directly proceed with adapter ligation.