NextSeq 500 paired end sequencing; Meiotic cohesin, chromatin and expression profiling of Arabidopsis thaliana floral buds from wild type and non-CG DNA methylation/H3K9me2 pathway mutant plants (ChIP-seq)
Meiotic cohesin, chromatin and expression profiling of Arabidopsis thaliana floral buds from wild type and non-CG DNA methylation/H3K9me2 pathway mutant plants (ChIP-seq)
Unopened flower buds at floral stage 9 were collected in tubes, snap frozen in liquid nitrogen and stored at -80°C until further use. Plants were grown under long day conditions (16 hours light/8 hours dark) at 20°C. Ten grams of unopened floral buds were ground in liquid nitrogen and used to perform ChIP-seq for SPO11-1 in wild type or for REC8 in wild type or kyp/suvh4 suvh5 suvh6 as follows: In vitro cross-linking and nuclei isolation were performed in nuclei isolation crosslinking buffer (60 mM Hepes pH 8.0, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 5 mM EDTA, 0.6% Triton X-100, 0.4 mM PMSF, 1 mM pepstatin, 1× protein cocktail inhibitor (Roche, 11873580001), 1% formaldehyde) at room temperature for 25 minutes. Glycine was added to a final concentration of 125 mM and the nuclei solution was incubated at room temperature for an additional 25 minutes. The nuclei were purified from cellular debris by filtering the nuclei solution twice through 1 layer of Miracloth and centrifugation at 12,000 g for 10 minutes at 4°C. The pellet was resuspended in EB2 buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 mM pepstatin, 1×protein cocktail inhibitor) followed by a centrifugation at 12,000g for 10 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in Nuclei Lysis Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 0.1 mM PMSF, 1 mM pepstatin, 1× protein cocktail inhibitor (Roche, 11873580001)). DNA was sonicated using a Bioruptor for 15 minutes (High intensity 320W, 30 seconds ON – 30 seconds OFF). The nuclei lysis solution was then diluted (3 volumes:2 volumes) into a solution of ChIP dilution buffer (1.1% Triton X-100, 1.1 mM EDTA, 20 mM Tris-HCl pH 8.0, 167 mM NaCl, 1 mM pepstatin, 1×protein cocktail inhibitor (Roche, 11873580001)) and a second cycle of DNA sonication was performed (25 minutes at high intensity 320W with 30 seconds ON – 1 minute OFF). The solution was then centrifuged at 5,000 g for 10 minutes at 4°C. 5% of the supernatant was kept as DNA input. The remaining supernatant was incubated with 10 μg of α-Myc (9E10, Santa Cruz Biotechnology - for SPO11-1), 7 μg of α-Myc (ab9132, Abcam – for REC8), or 7 μg α-HA (ab9110, Abcam – for REC8) antibodies that had been prebound to 50 μl of Dynabead Protein G (Invitrogen, 10004D) per gram of floral buds and was then incubated for 16 hours at 4°C. The immunopreciptated solution was washed 2 times for 5 minutes with low salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0) and 2 times for 5 minutes with high salt buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0). The proteins bound to the antibody/beads were treated with elution buffer (1% SDS, 0.1 M NaHCO3) at 65°C for 10 minutes. DNA-protein complexes were reverse crosslinked by adding NaCl to a final concentration of 240 mM and the solution was incubated at 65°C overnight. ChIP-DNA was purified with phenol/chloroform. 10 ng of ChIP-purified DNA was used to generate libraries using the TruSeq Library Preparation Kit v2 (Illumina), as described in Choi et al. (2018) Genome Res. 28(4): 532-546.
Experimental Factor: genotype
wild type genotype
Experimental Factor: immunoprecipitate
anti-HA (Abcam)