GSM4704522: HALO-GST_negative control; Vitis vinifera; OTHER
The genomic DNA was extracted from V. vinifera cv. Shiraz young leaves using a protocol adapted by Katsuhiro Matsui from Thomas et al., 1993.The leaves were harvested, frozen and ground in liquid nitrogen and stored at -80°C. Afterwards, 1g of powder was transferred to a 50ml falcon tube in which 12.5 ml of Crude Nuclei Buffer pH 7.6 (0.25M NaCl, 0.2M Tris-HCl pH 7.6, 2.5% PVP 40.000, 0.05M EDTA, 0.1% β-mercaptoethanol) were added. After a quick vortexing, the sample was centrifuged at 4°C for 10 minutes at 4000 rpm and the supernatant was discarded. The pellet was resuspended by vortexing with 5ml of DNA Extraction Buffer EtOH/Sark (0.5M NaCl, 0.2M Tris-HCl pH 8, 2.5% PVP 40.000, 0.05M EDTA pH 8, 3% N-Lauroylsarcosine, 20% EtHO,1% β -mercaptoethanol). The sample was left at 55°C for 30 minutes with frequent shacking and then added of 5ml chloroform. After vortexing, the solution was transferred into a 15 ml falcon tube and centrifuged at 20°C for 15 minutes at 5000 rpm. The upper phase was transferred into a new 50 ml falcon tube and 2.5 ml of isopropanol were added; the tube was mixed by inverting until a precipitate was seen and left at room temperature for 15 minutes. After a 15 minutes centrifuge at 5000 rpm and 20°C, the supernatant was poured off and the pellet was washed with 5 ml of 70% ethanol. The sample was re-centrifuged for 10 minutes at 5000 rpm and 20°C the pellet was air dried. Then, the dry pellet was resuspended in 600 µl of TE buffer (10mM Tris-HCl pH 8, 1mM EDTA pH 8) with 3 µl of RNase (stock 20mg/ml) and left overnight at 4°C. The solution was then transferred into a 1.5 ml collection tube and 300 µl of 7.5M NH4OAc were added. The DNA solution was centrifuged at room temperature for 10 minutes at 13.000 rpm, the supernatant was transferred into a new 1.5 ml tube and the DNA was precipitated with 0.5 ml of isopropanol. After another room temperature 10 minutes spin at 13.000 rpm, the pellet was washed again with 70% ethanol and let air dry. The DNA was finally resuspended in 300 µl of TE and its concentration was measured with the QubitTM dsDNA BR Assay kit (ThermoFisher). For the DNA library preparation, the gDNA was fragmented and ligated with a truncated Illumina TruSeq adapter as described in Bartlett at al., 2017 and Galli et al., 2018. 7.5 µg of gDNA were diluted in 130 µl of EB (Qiagen), loaded into a Covaris microTUBE and sonicated using a Covaris S2 sonicator to obtain the fragments peak size of 200 bp. The sheared sample was then transferred into a 1.5 ml low-bind tube, cleaned with AMPure XP beads (Beckman Coulter) at a 2:1 ratio of gDNA:beads and eluted in EB. The end-repair reaction was set up using the End-It kit (Lucigen) and the cleaning was made with the Qiaquick PCR Purification kit (Qiagen). The end-repaired fragments were A-tailed using the Klenow Fragment 3'-5' exo- (NEB) at 37°C for 30 minutes. After another Qiaquick purification, the truncated Illumina Y-adapter was ligated using the T4 DNA Ligase (NEB) at 16°C overnight. The enzyme was inactivated at 70°C for 10 minutes and then the sample was purified using a 1:1 ratio of library:beads. The final library was eluted in EB and quantified at the Qubit.