title: GSM8144938: suvh456_H3_ChIP_rep2; Arabidopsis thaliana; OTHER
library_construction_protocol: 5 g leaves were cross-linked in 1% formaldehyde for 10 min. Then stopped by adding 0.125 M glycine and washed by PBS. The nuclei were extrected by Honda buffer and washed 3 times by NRBT. Then the nuclei were sonicated 4 times for 10 min (30 s on/off intervals) using the Bioruptor® Pico. The sonicated nuclei were centrifuged at 15000rpm for 15 min, and the supernatant was transferred to new tubes and diluted 10 times with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl). 50 μL diluted supernatant was preserved as input. Then 2 μg anti-GFP antibody (Abcam, ab290) or anti-H3K9me2 antibody (Abcam, ab1220) was added into supernatant and rotate at 4 °C overnight. 50 μL washed Protein G Dynabeads (Invitrogen, 10004D) was added to the sample. After rotating for 4 h, the beads were washed sequentially with Low Salt Buffer, High Salt Buffer, LiCl Buffer, TE Buffer (each buffer for a quick wash, and a 10 min wash). Then add 100 μL 10% Chelex resin (Biorad, 1422842) to elute immune complexes by incubating at 95 °C for 10 min at 1300 rpm. After cooled to RT, 2 μL Protease K (10 mg/mL, Amresco 0706) was added into samples and incubated at 45 °C for 1 h, and then boiled at 95 °C and 1300 rpm for 10 min. Then the elution was purified with phenol-chloroform for sequencing. The library preparation steps were conducted according to the manual (ABclonal, RK20228). Briefly, the first adapter was ligated to the 3' end of the ssDNA using Adaptase via a highly efficient, proprietary reaction that only tails 3' ends of ssDNA and ligates the first truncated adapter to 3' ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequence-independent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter to the 5' ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified.