Protein a mag sepharose

A 50 ml culture-equivalent of yeast or 10 million cell-equivalent of K562 chromatin was diluted to 200 µl with IP Dilution Buffer and incubated overnight at 4 °C with the appropriate antibody. A 10 µl bed volume of IgG-Dynabeads was added to the yeast samples; and 3 µg of anti-CTCF antibody with a 10 µl slurry-equivalent of Protein A Mag Sepharose (GE Healthcare) was added to the K562 samples.

Proteins were extracted as described above for immunoblotting analysis, and protein concentrations were evaluated by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Protein A Mag Sepharose (GE Healthcare) was transferred to clean tubes and washed using a Protein A/G HP SpinTrap Buffer Kit and magnetic separation rack (GE Healthcare). After adding anti-EGFR, the samples were incubated with rotation for 1 h at room temperature, and the beads were separated from the lysate. Protein diluted with binding buffer was mixed with the beads, and the suspension was incubated with rotation at room temperature for 1 h. Then, elution buffer was added to the samples and the elution was collected.

Cells were harvested by trypsin-EDTA and fixed with 1% formaldehyde at room temperature for 10 min. After quenching the formaldehyde with 125 mM glycine, the cells were lysed with mammalian cell lysis buffer (Pierce, Thermo Fisher Scientific, Inc.) and treated with micrococcal nuclease (Fermentas, Thermo Fisher Scientific, Inc.) at 37°C for 20 min. The fragmented DNA solution was further diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl and 167 mM NaCl) and pre-cleared by incubation with 10 μl Protein A Mag Sepharose (GE Healthcare) at room temperature for 2 hours. After the pre-clear step, 1 g of anti-H3K27me3 antibody or normal rabbit IgG was added to the cell lysate, and the mixture was incubated at 4°C overnight. After washing, protein/DNA complexes were eluted using elution buffer (1% SDS and 100 mM NaHCO₃). Reverse crosslinking of the protein/DNA complexes was performed by treating with 200 mM NaCl at 65°C for at least 5 hours, and the proteins were digested with proteinase K. The DNA was further purified using a Wizard^TM PCR Clean-Up kit (Promega, Madison, WI, USA). The H3K27me3 binding region in the promoter of ZEB1 or NEUROD1 was further detected using SYBR Green quantitative PCR method by the primer sets described by Chaffer CL et al. [20 (link)].

Fungal genomic DNA was extracted as described above. After shearing with the Bioruptor sonicator (Diagenode), heat-denatured methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine (5-mC) (Active motif, # 39649) and Protein A Mag Sepharose (GE Healthcare, #28-9440-06) according to the manufacturer’s instructions. DNA SMART ChIP-Seq Kit (Clonetch) was used to construct a library for high-throughput sequencing on the MiSeq or HiSeq system (Illumina). Sequencing data were analyzed using Genomics workbench software v11.0 (CLCbio). For MeDIP mapping, the genome of the Magnaporthe oryzae strain 70-15 (release 8.0, http://www.broadinstitute.org/) was used as a reference sequence.

Co-Immunoprecipitation was carried out as described previously^{104 (link),105 (link)}. Infected insect cell membranes were dissolved in 1% CHAPSO-HEPES buffer (25 mM HEPES, pH7.4; 150 mM NaCl; 1% CHAPSO w/v; Buffer A) and diluted to 1 mg/ml protein concentration, using the same buffer. Soluble membranes were incubated with PS1 antibody Ab14 (rabbit polyclonal raised against 1–25 amino acids of PS1) overnight at 4 °C under gentle agitation. Protein-A Mag Sepharose (GE, UK 28951378) was used to harvest protein antibody complexes and eluted in LDS sample buffer (Invitrogen, USA NP0007) at 37 °C for 15 minutes for SDS-PAGE and Western immunoblotting.