title: MinION sequencing; Assembly of the Arabidopsis (Col-0) centromeres using Oxford Nanopore Technology long-read DNA sequencing.
design: Assembly of the Arabidopsis (Col-0) centromeres using Oxford Nanopore Technology long-read DNA sequencing.
library_construction_protocol: Plant were grown in long day growth conditions. 3 week-old Columbia (Col-0) seedlings grown on ½ MS media and 1% sucrose were harvested after keeping in the dark for 48 hours prior to harvesting.  For genomic DNA extraction, 3 week-old Columbia (Col-0) seedlings grown on ½ MS media and 1% sucrose and kept in the dark for 48 hours prior to harvesting. Approximately 10 g of tissue was used per 200 ml of MPD-Based Extraction Buffer pH 6 (MEB). Tissue was flash frozen and ground tissue in liquid nitrogen using a pestle and mortar and resuspended in 200 ml MEB. Ground tissue was thawed in MEB with frequent stirring. The homogenate was forced through 4 layers of miracloth, and then filtering again through 4 layers of fresh miracloth by gravity. 20% Triton x-100 was added to a final concentration of 0.5% on ice, followed by incubation with agitation on ice for 30 minutes.  The suspension was centrifuged at 800g for 20 minutes at 4oC.The supernatant was removed and the pellet reuspended using a paintbrush in 10 ml 2-methyl-2,4 pentanediol buffer pH 7.0 (MPDB). The suspension was centrifuged at 650g for 20 minutes at 4oC. The supernatant was removed and the pellet was washed with 10 ml of MPDB. Washing and centrifugation was repeated until the pellet appeared white and was finally resuspended in a minimal volume of MPDB. From this point onwards all transfers were performed using wide bore pipette tips. 20% SDS was added to a final concentration of 2% (w/v), mixed via gentle inversion and incubated at 60oC at 1 hour. The suspension was cooled to room temperature and 5M sodium perchlorate added to a final concentration of 1 M, followed by incubation on ice for 10 minutes. The lysate was then centrifuged at 20g for 20 minutes. The supernatant was transferred to a fresh tube. 5 ml CTAB buffer added to the nuclei pellet and mixed via gentle inversion, followed by incubation at 60oC until full lysis had occurred (between 30 minutes and 2 hours). An equal volume of chloroform was added and incubated on a rocking platform, with a speed of 18 cycles per minute, for 30 minutes, followed by centrifugation at 3000g for 10 minutes. An equal volume of phenol/chloroform/isoamyl alcohol (PCI, 25:24:1) was added to the lysate, followed by incubation on a rocking platform (18 cycles per minute) for 30 minutes. The lysate was centrifuged at 3000g for 10 minutes and the upper aqueous phase was transferred into a fresh tube. The PCI extraction was then repeated. The extraction was then repeated using only chloroform. 1/10th volume of 3M Sodium Acetate was added to the lysate and mixed by gentle inversion. Two volumes of ethanol were added and mixed by inversion. DNA was precipitated at -20oC for 48 hours. The precipitated DNA was removed using a glass hook and washed three times in fresh 70% ethanol. The DNA was dissolved in 120 µl of 10 mM Tris-Cl (pH 8.5).  Approximately 5 μg DNA was size selected for molecules greater than 30 kb using the BluePippin™ Size-Selection System (Sage Science) using the 0.75% DF Marker U1 cassette definition, with Range mode and BP start set at 30,000 bp. Library preparation was performed following the Nanopore SQK-LSK109 protocol and kit. Approximately 1.2-1.5 μg of size selected DNA in 48 μl was used for library preparation. DNA was nic-repaired and end-prepped by the addition of 3.5 μl of NEBNext FFPE Buffer and NEBNext Ultra II End Prep Reaction Buffer, followed by 2 μl of NEBNext DNA Repair Mix and 3 μl NEBNext Ultra II End Prep Enzyme Mix (New England Biolab, E7180S) with incubation for 30 min at 20°C followed by 30 min at 65°C. The sample was cleaned with 1×volume AMPure XP beads and eluted in 61 μl of nuclease-free water. Adapters were ligated at room temperature using 25 μl Ligation Buffer, 10 μl NEBNext T4 DNA Ligase and 5 μl Adapter Mix for 2 hours. The library was cleaned with 0.4×volume AMPure XP beads, washed using ONT Long fragment buffer and eluted in 15 μl elution buffer.