GSM7659083: Lm7210 WGS ONT; Lemna minor; WGS
High molecular weight (HMW) DNA extractions were performed using either a modified CTAB prep followed by a high-salt low-ethanol starch cleanup (Pacific Biosciences), or nuclear extraction as follows:  CTAB prep (Lg7742a, Lj8627, Wa8730): 4 g frozen tissue was ground three times under liquid N2 with a mortar and pestle and transferred to 20 mL pre-warmed lysis buffer (100 mM Tris-HCl pH 9.0, 2% w/v CTAB, 1.4 M NaCl, 20 mM EDTA, 2% PVP-10, 1% 2-mercaptoethanol, 0.1% sarkosyl, 100ug/mL Proteinase K), briefly vortexed, and then incubated at 65ºC for 1 hour with periodic mixing by gentle inversion. The lysate was extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) and then 1 vol. of chloroform:isoamyl alcohol in phase-lock gel tubes. HMW DNA was precipitated from the aqueous phase in a 50 mL falcon tube by the addition of 0.1 vol. 3M NaOAc pH 5.2 followed by 0.7 vol. Isopropanol. After gentle swirling but no inversion mixing, precipitated salt in the upper phase was pipetted off and HMW DNA was hooked out with a bent pasteur pipette, washed by dipping in 70% EtOH, air-dried for 2 minutes, and resuspended in 200 uL Tris-Cl pH 8.5 (EB). The DNA solution was treated with 2 uL 20 mg/mL RNase A at 37ºC for 20 minutes followed by 2 uL 50 mg/mL Proteinase K at 50ºC for 30 minutes. 194 uL EB, 100 uL 5M NaCl, and 2 uL 0.5 M EDTA were added and organic solvent extractions were performed as before (1x P:C:IA and 1x C:IA). 0.3 vol. EtOH was added followed by centrifugation at 16,000 x g for 15 minutes to precipitate carbohydrates. The supernatant was poured into a new tube and 1.7 vol. of EtOH was added to precipitate HMW DNA, followed by hooking with a pasteur pipette, a 70% EtOH wash, drying, and resuspension in 50 uL EB.  Nuclear prep (Lm7210, Lm9252, Lj7182, Lj9421, Lt9434): as previously described with modifications. Long-read data for Lg7742a, Lj8627, and Wa8730 were collected over multiple years as the MinION sequencing platform matured. Various protocols and both the SQK-LSK108 and SQK-LSK109 kits were used to prepare libraries for sequencing on multiple flow cell revisions. Here we describe the method used to produce the most recent runs in this study: 2 µg HMW DNA was sheared by 20 passes through a P1000 pipette tip and size-selected using 0.8 vol of custom SPRI beads. The SQK-LSK109 1D Genomic DNA Protocol was followed with the following modifications: Incubation times for all enzymatic reactions were extended to 20 minutes; cleanups following the repair reaction and adapter ligation were performed with 1 vol and 0.8 vol, respectively, of custom SPRI beads rather than Ampure XP; bead elutions were carried out at 37ºC until evenly resuspended; completed libraries were eluted in 24 uL Oxford EB, and half of this volume was mixed with LB and SQB and loaded onto an R9.4.1 rev. D flow cell. After about 20 hours the run was stopped, the Nuclease Flush protocol was performed, and the flow cell re-primed. 6 uL of the remaining library was mixed with 6 uL EB and the loading reagents, loaded onto the flow cell, and the run restarted.  Libraries for Lj7182, Lj9421, Lm7210, Lm9252, and Lt9434 were prepared as previously described and sequenced on R9.4.1 flow cells on the PromethION platform. Short read WGS libraries for Lg7742a and Lj8627 were prepared from 2µg of HMW gDNA using the Illumina TruSeq DNA PCR-Free kit (Illumina, cat#20015962) and sequenced on an Illumina MiSeq (PE 250bp, PE 300bp) or HiSeq 2500 instrument (PE 150bp). Libraries for other Lemna accessions were prepared as described for the NCBI SRA experiment SRX7624904, and previously published libraries were used for Wa8730 (SRX8008794, SRX8008795).