title: GSM6970366: ToCSV isolate from tolerant tomato line, 35 dpi, rep3; Tomato curly stunt virus; Bisulfite-Seq
library_construction_protocol: The leaf tissue was snap-frozen in liquid nitrogen and stored at -80 °C until needed. Total DNA was extracted from the harvested leaf tissue using the modified cetyltrimethylammonium bromide (CTAB) extraction method. Harvested snap-frozen leaf tissue was crushed to a fine powder using a micro pestle and resuspended in 500 µL of CTAB buffer (2% (w/v) CTAB, 5 M NaCl, 0.2% 2-mercaptoethanol, 0.5 M EDTA, 1 M Tris-HCI, pH 8.0 and 2% w/v PVP) and incubated at 65 °C for 60 min. For phase separation, 500µl of chloroform: isoamyl alcohol (24:1) was added, followed by centrifugation at 13 000 g for 10 min at room temperature. The aqueous phase was extracted to a new microfuge tube, and an equal amount of isopropanol was added to precipitate the DNA. The mixture was centrifuged at 13 000 g for 10 min, and the supernatant was removed. The pellet was washed twice in 1 ml of ice-cold 70% ethanol (v/v) followed by centrifugation at 13 000 g for 5 min. The pellet was air-dried and resuspended in TE buffer (1 M Tris, pH 8, and 0.5 M EDTA) supplemented with 200 µg/ml RNase A and stored overnight at 4°C. Total DNA was subjected to restriction digestion using the restriction endonuclease BamH1 (Thermo Scientific, Waltham, USA). The endonuclease digestion was carried out in a reaction that contained 1X FastDigest ® buffer (Thermo Scientific, Waltham, USA), 1.25 U/µL BamH1 enzyme and DNA template. The reactions were incubated at 37 °C for 16 hours and inactivated at 80 °C for 5 min. Followed by the addition of proteinase K (20mg/ml) and subsequent incubation for 15 min at room temperature and 10 min. Following the proteinase K treatment, the digested DNA was purified using the DNA clean-up (Zymo Research, Irvine, CA, USA). A total of 500 ng of purified DNA was used for bisulfite conversion, using the Zymo EZ DNA methylation Gold™ kit. Bisulfite-modified DNA was used as a PCR template using Epimark Taq polymerase (New England Biolabs). PCR was carried out in a final volume of 20 µL, containing, 1X Epimark buffer, 200 µM dNTPs, 0.2 µM primer, 0.0125 U/µL PCR of the EpiMark Hot Start Taq DNA Polymerase and 100 ng DNA template. PCR for each primer set was performed independently using the Eppendorf master cycler (Merck, US) with the following cycling conditions; initial denaturation 95 °C for 30 sec, followed by 40 cycles of denaturation 95 °C for 30 sec, annealing at specific temperatures for each primer set (see supplementary file) for 60 sec, extension 68°C for 1 min and a final extension at 68°C for 5 min. To confirm positive amplification, 5µL of the PCR amplicons were separated on a 1 % agarose gel stained with 10 µg/mL ethidium bromide and visualized under ultraviolet light, using a ChemiDoc MP imaging system (Bio-Rad, USA). TruSeq Nano (low molecular weight/adapter ligation)