title: GSM8508196: ChIP-seq_H3K9ac_ZT4biol rep2; Glycine max; ChIP-Seq
library_construction_protocol: Approximately 0.5 g of soybean mature leaves were used in each experiment. About 1 to 2g of the sample was ground into a fine powder in liquid nitrogen. Soybean tissues were crosslinked with 1% formaldehyde and quenched with 0.2 M glycine. For each experiment, about 1 to 2g of the sample was ground into a fine powder in liquid nitrogen. The nuclei were lysed in Honda buffer (0.44 M sucrose, 1.25% Ficoll,2.5% Dextran T40,20mM Hepes,10mM MgCl2, 0.5% Triton X-100, 5mM DTT, and protease inhibitor cocktail) at 4°C , and then the chromatin was fragmented into 200–600 bp by ultrasound processing using a Bioruptor (Diagenode). The lysates were centrifuged at 16000 g at 4 °C for 20 min. Then 2-5μL antibody (H3K9ac,) and 40μL suspended protein G magnetic beads (Life Technologies) were incubated at 4°C for 8h and rotated. The protein-DNA complexes were subjected to immunoprecipitation by incubating the antibody-bead complexes with the fragmented chromatin. Then, the target protein-DNA complexes were eluted from the beads by adding 1mL of freshly prepared High salt buffer, low salt buffer, LiAc buffer, and TE buffer. After reverse cross-linking of DNA and protein by Chelex-100, ChIP-DNA was extracted using phenol: chloroform: isoamyl alcohol. ChIP-DNA libraries were prepared using a NEBNext ®Ultra ™II DNA Library Preparation Kit. DNA clean beads (vazyme, N411) were used to select 250–650 bp library fragments.