title: GSM7310404: WGBS data of suvh2/9, replicate 2; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: Genomic DNA for WGBS was extracted with DNeasy Plant Mini Kit (QIAGEN). Genomic DNA for BS-PCR-seq was extracted with CTAB method. RNA was extracted with Direct-zol RNA MiniPrep kit (Zymo Research). ChIP-seq library preparation: 15 ml of unopened flower buds were collected for each ChIP and flash-frozen in liquid nitrogen. The flower tissue was ground to fine powder with Retsch homogenizer in liquid nitrogen and resuspended in nuclei extraction buffer (50 mM HEPES pH 8.0, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 0.6% Triton X-100, 0.4 mM PMSF, 5 mM benzamidine, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), 50uM MG132). For transgenic lines of MOM1-MYC and PIAL2-MYC, EGS was first added to resuspended lysate to 1.5 mM and the tissue lysate was incubated at room temperature for 10 min with rotation. Then the lysate was supplemented with formaldehyde at 1% and rotated at room temperature for another 10 min followed by adding glycine to stop crosslinking. For ChIP of all other proteins, crosslinking was performed by directly supplementing formaldehyde to 1% without adding EGS, then rotated at room temperature for 10 min followed by adding glycine to stop crosslinking. The crosslinked nuclei were isolated, lysed with Nuclei Lysis Buffer and diluted with ChIP Dilution Buffer. Then the lysate was sonicated for 22 min (30 s on/30s off) with Bioruptor Plus (Diagenode). After centrifugation, antibody for FLAG epitope (M2 monoclonal antibody, Sigma F1804, 10 ul per ChIP) or for MYC epitope (Cell Signaling, 71D10, 20 ul per ChIP) were added to the supernatant and incubated at 4 ℃ overnight with rotation. Then, Protein A and Protein G Dynabeads (Invitrogen) were added and incubated at 4 ℃ for 2 hours with rotation. After that, the beads were washed and eluted, and the eluted chromatin was reverse-crosslinked by adding 20 ul 5 M NaCl and incubating at 65 ℃ overnight followed by treatment of Proteinase K (Invitrogen) for 4 hours at 45 ℃. DNA was purified and precipitated with 3 M Sodium Acetate, GlycoBlue (Invitrogen) and ethanol at -20 ℃ overnight. After centrifugation, the precipitated DNA was washed with ice cold 70% ethanol, air dried and dissolved in 120 ul of H2O. ChIP-seq libraries were prepared with Ovation Ultra Low System V2 kit (NuGEN). RNA-seq library preparation: Twelve-day old seedlings grown on half MS medium (Murashige and Skoog Basal Medium) were collected and flash-frozen in liquid nitrogen. RNA was extracted with Direct-zol RNA MiniPrep kit (Zymo Research) and 1ug of total RNA was used to prepare RNA-seq libraries with TruSeq Stranded mRNA kit (Illumina) WGBS library preparation: Rosette leaves of about one-month-old Arabidopsis plants were collected for DNA extraction using DNeasy Plant Mini Kit (QIAGEN). 500 ng DNA was sheared with Covaris S2 (Covaris) into around 200bp at 4℃. The DNA fragments were used to perform end repair reaction using the Kapa Hyper Prep kit (Roche), and together with Illumina TruSeq DNA sgl Index Set A/B (Illumina) to perform adapter ligation. The ligation products were purified with AMPure beads (Beckman Coulter), and then converted with EpiTect Bisulfite kit (QIAGEN). The converted ligation products were used as templates, together with the primers from the Kapa Hyper Prep kit (Roche) and MyTaq Master mix (Bioline) to perform PCR. The PCR products were purified with AMPure beads (Beckman Coulter) and used for sequencing. ATAC-seq library preparation: About 5 grams of unopened flower buds was collected and immediately transferred into ice-cold grinding buffer (300 mM sucrose, 20 mM Tris pH 8, 5 mM MgCl2, 5 mM KCl, 0.2% Triton X-100, 5 mM β-mercaptoethanol, and 35% glycerol). The samples were ground with Omni International General Laboratory Homogenizer on ice and then filtered through a two-layer Miracloth and a 40-µm nylon mesh Cell Strainer (Fisher). Samples were spin filtered for 10 min at 3,000 g, the supernatant was discarded, and the pellet was resuspended with 25 ml of grinding buffer using a Dounce homogenizer. The wash step was performed twice in total, and nuclei were resuspended in 0.5 ml of freezing buffer (50 mM Tris pH 8, 5 mM MgCl2, 20% glycerol, and 5 mM β-mercaptoethanol). Nuclei were subjected to a transposition reaction with Tn5 (Illumina). For the transposition reaction, 25 µl of 2x DMF (66 mM Tris-acetate pH 7.8, 132 mM K-Acetate, 20 mM Mg-Acetate, and 32% DMF) was mixed with 2.5 µl Tn5 and 22.5 µl nuclei suspension at 37°C for 30 min. Transposed DNA fragments were purified with ChIP DNA Clean & Concentrator Kit (Zymo). Libraries were prepared with Phusion High-Fidelity DNA Polymerase (NEB) in a system containing 12.5 µl 2x Phusion, 1.25 µl 10 mM Ad1 primer, 1.25 µl 10 mM Ad2 primer, 4 µl ddH2O, and 6 µl purified transposed DNA fragments. BS-PCR library preparation: Rosette leaves of about one-month-old plants were collected and subject to DNA extraction with CTAB method followed by bisulfite DNA conversion using the EpiTect Bisulfite kit (QIAGEN) kit. Three regions of the FWA gene were amplified from the converted DNA with Pfu Turbo Cx (Agilent): Region 1 (chr4: 13038143-13038272), Region 2 (chr4: 13038356- 13038499) and Region3 (chr4: 13038568-13038695). Libraries were prepared with the purified PCR product by the Kapa DNA Hyper Kit (Roche) together with TruSeq DNA UD indexes for Illumina (Illumina) and were sequenced on Illumina iSeq 100 or HiSeq 4000 instruments.