GSM9100198: Input wild-type Tak1, 14d thalli, biol rep1 (Input); Marchantia polymorpha; ChIP-Seq
Two-week-old thalli started from gemmae of M. polymorpha Tak-1 wild type were collected and cross-linked using 1% formaldehyde in 1× PBS under vacuum on ice for 10 min. The cross-linking reaction was quenched by adding 2 M glycine to achieve a final concentration of 0.125 M under vacuum on ice for 10 min. Excess solution was removed from cross-linked tissue by blotting with paper towels. Cross-linked tissue was then snap frozen in liquid nitrogen and ground to a fine powder using mortar and pestle. The powder was transferred into a 50-ml plastic tube and suspended in 40 ml of MP1 buffer (10 mM MES-KOH buffer pH 5.3, 10 mM NaCl, 10 mM KCl, 0.4 M sucrose, 2% (w/v) PVP-10, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 6 mM EGTA, 1× cOmplete protease inhibitor cocktail). Suspended samples were then filtered twice through one layer of Miracloth, once through a 40 μm nylon mesh, and twice through a 10 μm nylon mesh. Filtered samples were centrifuged at 3000 × g at 4°C for 10 min, and the supernatant was discarded. The pellet was washed using 15 ml of MP2 buffer (10 mM MES-KOH buffer pH 5.3, 10 mM NaCl, 10 mM KCl, 0.25 M sucrose, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.2% Triton-X 100, 1× cOmplete protease inhibitor cocktail) three times. The final pellet was then resuspended in 5 ml of MP3 buffer (10 mM MES-KOH buffer pH 5.3, 10 mM NaCl, 10 mM KCl, 1.7 M sucrose, 2 mM MgCl2, 10 mM 2-mercaptoethanol, 1× cOmplete protease inhibitor cocktail) and centrifuged at 16 000 × g at 4°C for 1 h. After centrifugation, the supernatant was discarded, and the pellet was resuspended in 900 μl of covaris buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris–HCl pH 8.0, 1× cOmplete protease inhibitor cocktail). The resuspended pellet containing the chromatin fraction was fragmented using a Covaris E220 High-Performance Focused Ultrasonicator for 15 min at 4°C (duty factor, 5.0; peak incident power, 140.0; cycles per burst, 200) in a 1-ml Covaris milliTUBE. Sheared chromatin was centrifuged at 20 000 × g at 4°C for 10 min, and the supernatant was transferred into a new 5-ml tube and diluted by adding 2.7 ml of ChIP dilution buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7mM Tris-HCl pH 8.0, 167 mM NaCl). Diluted chromatin was cleared by incubating with proteinA/G beads (Thermo Fisher Scientific, Waltham, MA, USA) at 20 rpm spinning at 4°C for 1 h. Beads were removed by magnetic racks and precleared chromatin was separated into five tubes and incubated with 1 μg of specific antibodies for histone H2A variants () at 20 rpm spinning at 4°C overnight. Chromatin bound by antibodies was collected by incubating with protein A/G beads for 3 h. The beads were collected by magnetic racks and washed twice with a low salt wash buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 0.1% SDS), once with a high salt wash buffer (20 mM Tris–HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 0.1% SDS), once with a LiCl wash buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% IGEPAL CA-630 and 0.1% sodium deoxycholate), and twice with a TE buffer (10 mM Tris–HCl pH 8.0 and 1 mM EDTA). Immunoprecipitated DNA was eluted using 500 μl elution buffer (1% SDS and 0.1 M NaHCO3) at 65°C for 15 min. To reverse cross-link, eluted DNA was mixed with 51 μl of reverse cross-link buffer (40 mM Tris–HCl pH 8.0, 0.2 M NaCl, 10 mM EDTA, 0.04 mg ml−1 proteinase K; Thermo Fisher Scientific) and incubated at 45°C for 3 h and then at 65°C for 16 h. After cross-link reversal, DNA was treated with 10 μg of RNase A (Thermo Fisher Scientific), incubated at room temperature for 30 min and purified using the MinElute PCR purification kit (Qiagen). ChIP-seq library was generated from ChIPed DNAs using Ovation® Ultralow Library Systems V2 (Tecan, Männedorf, Switzerland).