title: 2D_Trypsin_Resin_rSAP_Kinase_Ligase_Barcode113_FLO-MIN106
design: Total genomic DNA was extracted from blood using the Qiagen Blood & Cell Culture DNA Mini Kit (Hilden, Germany, cat# 13323), yielding 150 ng/ul provided in TE buffer. Sequencing was undertaken using the Oxford Nanopore MinIon (machine ID No. MN19030; flow-cell version FLO-MIN106)  with R9.4 chemistry and conducted using Minion protocol version GDE_9002_v108_revT_18Oct2016 for library preparation with modification. A nick-repair enzyme (NEBNext FFPE Repair Mix) to repair breaks in one side of the DNA double helix, which can prematurely shorten DNA reads was added. To increase library yield, NEBNext Ultra II End Repair/dA-Tailing Module was used to eliminate an additional SPRI (Solid Phase Reversible Immobilization) cleanup step, here applying Agencourt AMPure XP beads. To increase library yield, 2 ul of concentrated NEB T4 DNA ligase was added during adaptor ligation.1. DNA Nick-Repair, Blunt-Ending, and End Repair Add in a clean low bind PCR tube, (a) ~ 2 ug genomic Tuatara DNA as described above of 8.27 ul, (b) NEBNext FFPE Repair Mix of 3 ul, (c) NEBNext Ultra II End Repair/dA-Tailing Mix of 3 ul, (d) NEBNext Ultra II End Repair/dA-Tailing Buffer of 7 ul, (e) 100X NAD+ of 0.6 ul, and (f) 10 mM Tris HCl pH 8.5 buffer of 38.13 ul for a total volume of 60 ul. This is mixed gently via inversion and incubated at 20 C for 60 min and 65 C for 30 min in a thermocycler. An additional step (g) added 3 ul of NEB Antartic Phosphatase and 6 ul of NEB Antartic Phosphatase buffer containing Zn2+ for enzymatic activation and is also mixed gently via inversion and incubated at 37 C for 30 min in a thermocycler.2. Immobilized Trypsin-Resin in Place of SPRI Cleanup For this run a deviated protocol to increase library yield from wash cycles and avoid unnecessary DNA shearing from agitation, applied an enzymatic digest of end-repair enzymes in step 1 above replacing the use of SPRI beads. This was done using an activated trypsin. 2.1. To activate trypsin resin, resin was gently mixed adding 20 ul of resin to 100 ul of 10 mM Tris HCl pH 8.5 buffer in a 1.5 ml tube. This was inverted for mixing flowed by a 6000 rpm spin, with the supernatant discarded; this was repeated two additional times, with 20 ul of 10 mM Tris HCl pH 8.5 buffer added to the end product.2.2. The (a) end-repaired DNA from step 1 of 60 ul, and (b) activated trypsin resin of 20 ul from step 2.1 were (c) incubate at 37 C for 30 min. Only 60 ul of the total volume of 80 ul was retained leaving 20 ul of pelleted resin behind.3. An additional step added  (a) 2 ul of T4 Polynucleotide Kinase, (b) 10 ul of T4 Polynucleotide Kinase buffer, and 10 ul of 10 mM ATP. This is mixed gently via inversion and incubated at 37 C for 30 min in a thermocycler.4. 2D Adaptor Ligation Add to (a) eluted End-prepped DNA above of 30 ul, (b) 2D Adapter Mix of 10 ul, (c) dH2O of 5 ul, (d) HP Adapter of 2 ul with (d) NEB Instant Sticky End Ligase of 50 ul and (e) incubated for 10 min at room temp; followed with (f) HP tether of 1 ul and (g) 2 ul of concentrated NEB T4 DNA ligase added with (h) additional incubation at room temp for 10 min. The 2 ul of concentrated NEB T4 DNA ligase was added during adaptor ligation with the HP Tether to increase library yield. The total yield is 100 ul for this step.5. Library Purification requires two steps.5.1. MyOne C1 Streptavidin beads preparation Add in a 1.5 ml Eppendorf DNA LoBind tube (a) MyOne C1 Streptavidin beads of 50 ul and (b) pellet beads on magnet for 2 min; (c) discard supernatant and (d) add Oxford Nanopore Bead Binding Buffer of 140 l; (e) vortex until homogeneous and (f) pellet on a magnet for 2 min; (g) discard supernatant and (h) repeat Oxford Nanopore Bead Binding Buffer wash step of 140 ul with pelleting on a magnet for 2 min X 2; (i) add Bead Binding Buffer of 100 ul and (j) label tube as Washed Beads for binding step.5.2. Purification of Library using Streptavidin Beads For binding add (a) Washed Beads from previous step 5.1 of 100 ul to the tube containing the Ligated DNA in step 4 of 100 ul and (b) incubate at room temperature for 5 min; for elution add (c) Oxford Nanopore elution buffer to DNA-bound beads of 25 ul, (d) incubate tube on hot block at 37 C for 10 min, (e) pellet beads on a magnet for 2 min, and (f) transfer supernatant containing library into a clean 1.5 ml Eppendorf DNA LoBind tube for DNA sequencing.Library was run on a FLO-MIN106 flow-cell.