GSM6807211: TM Rec7_7 met; Triticum timopheevii subsp. timopheevii; Bisulfite-Seq
DNA was extracted from 100 mg lyophilized leaf segments disrupted with two 5 mm glass beads in sealed 96-well 2ml plates. Lysis was performed in 1 ml of lysis buffer at 65°C for 45 min. Subsequently, plates were centrifuged at 1300 × g for 10 min at 4°C. DNA purification was done at a Biomek® NXP automated workstation. A 100 µl of lysate was transferred to a new PCR plate and mixed with 70 µl of isopropanol and 5 µl of Agencourt® Genfind® v2 magnetic beads and incubated for 5 min at RT. Subsequent steps comprised 5 min incubation at the magnet, supernatant discarding, and three-time wash with 100 µl of 70% ethanol. Elution was done in 100 µl of H20 with 10 min incubation at RT and 10 min incubation at the magnet. Eluted DNA was transferred to a new PCR plate. DNA methylation in the regions with recombination events was determined using the EZ DNA Methylation-Gold Kit (Zymo Research, USA). 400 ng of genomic DNA was bisulfite converted and purified according to the manufacturer's instruction (ver. 1.0.1). For amplification of the bisulfite converted DNA, a set of new primers was designed. For amplification and sequencing of the Hotspot region part 1 after CT-conversion we used: Hot1_Met_F 5'-TTGTGTGTGTAGTTAGGTATAAAAAGT-3' and Hot1_Met _R 5'-ACTTCTTCTCCATACATCCTAATC-3' For amplification and sequencing of the Hotspot region part 2 after CT-conversion we used: Hot2_Met_F 5'- TTGTTTATTTTTTTTATTTAGTGT -3' and Hot2_Met _R 5'-AAACRAATACCAAAATTTTTCATTCTCTAT-3' For amplification and sequencing of the Hotspot region part 3 after CT-conversion we used: Hot3_Met_F 5'- TTAAATTTTTAAAGAAGATAAAGAGAG -3' and Hot3_Met _R 5'- ATATTCCTCCATCACCATCACACTAC -3' For amplification and sequencing of the Hotspot region part 4 after CT-conversion we used: Hot4_Met_F 5'- TAATAAGATGYGTTGGGTAGAGGTG -3' and Hot4_Met _R 5'- CTTCTAAACRCCATTAAAAACTTCATTA -3' For amplification and sequencing of the Rec7 region part 1 after CT-conversion we used: Rec7_1F_Met 5'- GAATGTTATTTGTTAGTAGA -3' and Rec7_1R_Met 5'- AAACTAAAAACTTTAAAACTTATCAT -3' For amplification and sequencing of the Rec7 region part 2 after CT-conversion we used: Rec7_2F_Met 5'- TAGTATGAAATATGATATGTTTTT -3' and Rec7_2R_Met 5'- ATAAATACAACTCAACCAAC -3' For amplification and sequencing of the Rec7 region part 3 after CT-conversion we used: Rec7_3F_Met 5'- AAAGATAGTAATAGAAGTAATAAGT -3' and Rec7_3R_Met 5'- TAAAACCTTACAATCCAACA -3' For amplification and sequencing of the Rec7 region part 4 after CT-conversion we used: Rec7_4F2_Met 5'- TTTGAGTTGTTGTTATAATT -3' and Rec7_4R4_Met 5'- CTTCACCAACRACTCCAA -3' For amplification and sequencing of the Rec7 region part 5 after CT-conversion we used: Rec7_5F4_Met 5'- ATTTAATAAAGTTTAGAGTTYG -3' and Rec7_5R3_Met 5'- ACACCCRCCATACCTTAA -3' For amplification and sequencing of the Rec7 region part 6 after CT-conversion we used: Rec7_6F_Met 5'- TTTGTTAAAAATTAAGGTATGG -3' and Rec7_6R_Met 5'- ATATTTTCATACCTAAATCACATAAA -3' For amplification and sequencing of the Rec7 region part 7 after CT-conversion we used: Rec7_7F_Met 5'- TGAAGAAAATTTTGAAAAATATGAA -3' and Rec7_7R_Met 5'- ATTCCRACAATTTATTTTTA -3'