GSM8699309: MBD5_input_rep1; Solanum lycopersicum; ChIP-Seq
Dneasy®  Plant  Maxi Kit (Qiagen) The ChIP-seq experiment was performed as described in Zhao et al (2020). Young leaves were crossed with 1% formaldehyde in phosphate-buffered saline (PBS) to fixed DNA and protein. Then 0.2M glycine was used to stop the fixation, then washed with ddH2O for 3 times and freeze in liquid nitrogen. About 0.4g samples were ground in liquid nitrogen and resuspend in EB1 buffer in 2.4ml. The mixture was mixed in 4°C for 15mins. Samples were sonicated for 20 cycles firstly and then centrifuged at 10000xg for 10min at 4°C, and the supernatant was collected for the following 45 cycles to fragmented into 200-600bp. The chromatin samples was then took out and saved 50ul supernatant as input for subsequent analysis. 50 μl of Dynabeads® protein G beads (Invitrogen, 10003D) per sample were washed with 300 μl of PBST buffer (PBS with 0.1% Tween 20) twice and resuspended in 200 μl of PBST buffer. Then antibody (anti-flag, 5-10 μl) was added to the beads and incubated for about 4-6 h on a rotator at 4 °C to form antibody coated beads . Next step, The following samples was incubate with pre-cleared beads for 1-3h at 4 °C , and then washed with PBST twice. IP products was incubated with antibody coated with protein G for overnight at 4 °C on a rotator at 4 °C to immunoprecipitated the target chromatin. In the next day, beads were collected and washed with ice cold low-salt ChIP buffer (50mM HEPES-KOH, 150Mm Nacl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate and 0.1% SDS) twice, high Salt buffer (NaCl in low salt ChIP wash buffer was replaced with 350mM NaCl) twice; ChIP wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA, 0.1% sodium deoxycholate) once, and TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) twice. Then protein -DNA was eluted twice with 100ul and 200ul ChIP Elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) ;After that, 5 μl of proteinase K and 6μl of 5M NaCl was added to the elute and incubated for 6 h or overnight at 55 °C to reverse crosslinking the protein-DNA complexes. Then, ChIP DNA was purified with DNA clean Kit＆concentrator kit from ZYMO Research. ChIP DNA library was built with NEB Next Ultra II DNA library prep kit for Illumina sequencing.