title: GSM5194401: H3K9me2 - suvh2suvh9+SUVH9(Y636F) R2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: Siliques were cut open with insulin syringe needles, whole seeds were transferred to a tube and frozen. Fixed chromatin was isolated from approximately 250 to 300 whole seeds using a scaled-down version of a protocol found elsewhere. Briefly, seed samples kept at -80 °C are thawed on ice in 1 % formaldehyde. Vacuum is applied for 10 min before adding glycine to a final concentration of 0.125 M and vacuum is applied for another 5 min. Embryos are collected at the bottom of the 1.7 mL tube by spinning 1 min at 5000 rpm and washed three times with distilled water. Once last wash is removed, tubes are put in liquid nitrogen and embryos are ground to a fine powder with pre-cooled plastic pestle and 750 uL of extraction buffer I (0.4 M Sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 25 mM β-mercaptoethanol, 1X Halt™ Protease Inhibitors (Thermo Fisher Scientific)  and 0.1 mM PMSF). Slurry is transferred and processed in a 2 mL Dounce homogenizer (MilliporeSigma) and transferred to a 15 mL conical tube on ice. When all samples are processed, tubes are inserted in a mixer slowly rotating at 4 °C form 30 min. The slurries were then filtered through a 40 µm FalconTM cell strainer (Thermo Fisher Scientific)  into a new 50 mL FalconTM tubes and tubes and filters were rinced with 5 mL of buffer I. The new tubes were spun at 4000 rpm for 20 min at 4 °C. Supernatants are poured off and pellets are resuspended in 1.25 mL of extraction buffer II (0.25 M Sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 1X Halt™ Protease Inhibitors (Thermo Fisher Scientific)  and 0.1 mM PMSF) and transferred to a new DNA Lobind 1.75 mL tubes (Eppendorf). Tubes are spun at 13,000 rpm for 10 min at 4 °C. Supernatants are discarded and pellets are resuspended in 350 µL of buffer II with repeated pipetting. The solutions are then added to new tubes on top of a 350 µL of buffer III (1.7 M Sucrose, 10 mM Tris-HCl pH 8.0, 2 mM MgCl2, 0.15% Triton X-100, 5 mM β-mercaptoethanol, 1X Halt™ Protease Inhibitors and 0.1 mM PMSF). Tubes are spun at 13,000 rpm for 45 min at 4 °C. Remove supernatants and resuspend pellets in 70 µL of Nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1X Halt™ Protease Inhibitors and 0.1 mM PMSF) with repeated pipetting. Incubate tubes on ice for 30 min. Shear chromatin using a Covaris® S220 ultrasonicator at a frequency of 175 MGh for 6 min. Tubes are spun at 13,000 rpm for 10 min at 4 °C, supernatants (circa 60 µL) are transferred to new tubes.The following procedures are executed in the cold room unless indicated otherwise. 540 µL of ChIP buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 1.1% Triton X-100, 167 mM NaCl and 1X Halt™ Protease Inhibitors) is added to the sheared chromatin and 20 µL of pre-washed Dynabeads™ Protein A (Thermo Fisher Scientific)  is added for a 1 hour pre-clearing with rotation. Beads are separated with magnet and supernatants are transferred to new tubes. Solutions are split equally between two new tubes. 2.5 µg of antibody is added to each tube, either anti-Histone H3 di-methyl (ab1220 from Abcam) or anti-Histone H3 unmodified  (ab1791). 25 µL of pre-washed beads are added to each tube and they are incubated overnight with rotation. Beads are collected and washed consecutively with 1 mL low salt buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100,, 0.1% SDS and 150 mM NaCl), 1 mL high salt buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 500 mM NaCl), 1 mL LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate and 250 mM LiCl) and 1 mL TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) 5min each with rotation. TE wash is repeated then beads are eluted twice with 125 µL of elution buffer (1% SDS and 100mM of NaHCO3) with vortexing and 15 min incubation at 65 °C. After combining the two elutions, 10 µL of 5 M NaCl is added and solutions are incubated at 65 °C overnight to reverse cross-linking. 10 µL of 500 mM EDTA, 20 µL of 1 M Tris-HCl pH 6.5 and 1 µL of 20 mg/mL Proteinase K are added and solutions are incubated at 45 °C for 1 hour. DNA is then purified using Zymo Research ChIP DNA Clean and Concentrator columns eluted twice with 25 µL of 10 mM Tris-HCl pH 8.0. DNA fragments are made into libraries using the NEBNext Ultra II FS kit (New England Biolabs) according to manufacturer's instructions. Libraries were pooled together.