title: Nanopore sequencing of xenic cultures of Epithemia clementina
design: High molecular weight DNA extraction   E. clementina were grown to a density of approximately 400,000 cells/mL and 20-30 million cells were used as input to HMW DNA extraction. Xenic cultures were first subjected to a round of centrifugation through a discontinuous Percoll gradient consisting of 10mL layers of of 80%, 70%, 60% and 50% v/v percoll stock solution (90% Percoll and 10% 1.5M NaCl) and CSi-N media. E. clementina cells pellet out of the solution entirely, whereas a portion of their bacterial community stays suspended in various Percoll fractions. Centrifugation steps were performed at 23C at 1000 x g. For both xenic and axenic cultures, HMW DNA was isolated using a nuclei extraction method adapted from64. Cells were suspended in a minimal volume of nuclear isolation buffer (NIB, detailed components in64) and then transferred to a mortar where they were flash frozen with liquid nitrogen and ground with the pestle until a paste formed. This grinding process was repeated a total of three times. Cell homogenate was transferred to a 15mL falcon tube containing NIB, rinsing the mortar with NIB if necessary, and incubated at 4C for 15min. No miracloth filtering step was performed. The cell homogenate was spun down at 4C and 2900 x g. The resulting nuclei pellet was rinsed with 15mL NIB until the solution was clear of any photosynthetic pigments. The resulting nuclei/cellular compartment mix was used as input for the Nanobind plant nuclei big DNA kit from PacBio. Steps were followed as listed in the kit protocol except for large cell inputs, in which reagent volumes were doubled and the Proteinase K digestion step was extended to 2hrs. The isolated DNA from this protocol was processed with the Short Read Eliminator kit from PacBio to deplete DNA fragments < 25kb in length. The final, HMW DNA was used as input for nanopore library preparation.  Nanopore library preparation and sequencing   For all sequencing runs from axenic cultures of E. clementina, 1-2g of HMW DNA was used as input to the Oxford Nanopore Technology sequencing by ligation kit (SQK-LSK112). The nanopore protocol (Version: GDE_9141_v112_revC_01Dec2021) was followed with the following minor modifications: end repair incubation was lengthened to 30min at 20C and the adapter ligation incubation was lengthened to 60min at room temperature. Resulting libraries were loaded onto primed, high-accuracy MinION R10.4 flow cells (FLO-MIN112) at a target amount of 9 fmoles of 10kb DNA. All sequencing of xenic cultures was performed similarly, but with previous iterations of the sequencing kit (SQK-LSK110) and the flow cell MinION R9.4.1 (FLO-MIN111). If pore occupancy dipped below roughly 1/3 of starting occupancy during the sequencing run, the run was paused, and the flow cell was washed with the Flow Cell Wash Kit (EXP-WSH004) from Nanopore according to the associated protocol. The same prepped library was then reloaded onto the flow cell and the sequencing run was restarted. Each run was left to sequence for 3-5 days, or until pore occupancy was near zero.