GSM8224216: N_gaditana_Dark_3; Nannochloropsis gaditana; Bisulfite-Seq
Whole genome bisulfite sequencing was performed as previously described (Steadman 2020). Genomic DNA from N. salina and N. gaditana was isolated from three sets of cultures: nitrogen replete (full nutrients three days prior to nitrogen starvation), nitrogen starvation (the day nitrogen was not detectable in the media), and nitrogen depletion (three days after nitrogen starvation occurred). One ug of gNDA was digested with 1 unit of dsDNA Shearase Plus (Zymo catalog #E2018-50). Fragments were end-blunted, 3'-terminal-A extended, and purified using DNA Clean & Concentrator kit (Zymo catalog #D4003). Fragments were ligated to pre-annealed adapters containing methylated (5'-methyl) cytosine, filled in, and treated with sodium bisulfite using the EZ DNA Methylation-Lightning kit (Zymo catalog #D5003) . Illumina TruSeq indices were used for PCR; bisulfite treated fragment size and concentration were determined using an Agilent 2200 TapeStation (Agilent catalog #G2991BA). Fragments were sequenced on an Illumina HiSeq 2500.2x108 N. gaditana cells were processed using the Qiagen HW MagAttract kit (Qiagen catalog #67563 ) following the protocol for “gram (-) bacteria”. Prior to processing, cells were resuspended in buffer ATL and lysed by eight freeze thaw cycles -- 5 minutes on dry ice (-37c), followed by 5 minutes in a heat block at (37c) . DNA was eluted in 190uL ddH20 to prevent carryover of PCR inhibitors. To remove remaining RNA not removed by the kit, 400ug (4uL: 10mg/mL) RNASE A (ThermoFisher catalog #EN0531) was added with incubation at 37c for 1hr. Subsequently, to reduce polysaccharide contamination, 200uL 2X CTAB buffer was added to DNA solution (1.4 M NaCl, 20mM EDTA: pH 8.0, 2% CTAB w/v, 0.2% v/v β- mercaptoethanol, 100 mM Tris-Cl: pH 8.0), 400uL 25:24:1 Phenol:Chloroform:Isoamyl Alcohol buffered to pH 8.0 (Sigma Aldrich catalog #P2069) added, solution inverted 40X and phases separated by 5 minutes of centrifugation at 5000g. The aqueous phase was retained, taking care to avoid carryover of lipids or polysaccharides in the interphase. DNA was precipitated at -80c for 1hr by addition of 1/10 volume of NaACOH and ice-cold Ethanol (200 proof) (Thermo Fisher catalog #T036181000) to a final concentration of 75%, pelleted by centrifugation for 15 minutes at 7500g, washed with ice cold 75% ethanol, repelleted by 15 minutes centrifugation at 7500g, and air dried in the hood. DNA was resuspended in nuclease-free ddH20 and quantified using the Qubit 3.0 fluorometer and gDNA tapestation kit (Agilent catalog #5067-5366). WBGS libraries were prepared using zymo kit #D5003. ONT libraries were constructed by: Libraries were prepared using the Native Barcoding DNA Ligation Kit K14 from nanopore (ONT catalog # SQK-NBD114.96). An E. coli DNA methylation null control was also prepared using the above ONT kit (Zymo catalog #D5016). Libraries were loaded onto a V11.4 Promethion flow cell (ONT catalog #FLO-PRO114M) with light shield (ONT catalog #SPR-FLS001P) and sequenced using MinKNOW with real-time basecalling set to fast and minimum length set to 200bp. Bisulfite-seq and Oxford nanopore