GSM7659056: Lj8627 WGBS 1; Lemna japonica; Bisulfite-Seq
High molecular weight (HMW) DNA extractions were performed using a modified CTAB prep followed by a high-salt low-ethanol starch cleanup (Pacific Biosciences) as follows:  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. WGBS libraries were prepared for Sp9509, Lg7742a, Lj8627 and Wa8730 as described previously (Parent et al. 2021) for Arabidopsis embryos, except that DNA from the HMW extraction methods described above was used. Libraries were sequenced on a NextSeq 500 (PE 151) or a HiSeq 2500 (PE 108)