title: GSM5418477: TM-1-leaf-K4me3_rep2_clean; Gossypium hirsutum; ChIP-Seq
library_construction_protocol: 20 d-old cotton leaves were used for nuclei preparation. The nuclei were extracted using nuclei isolation buffer (NIB, pH 5.0, 1.0 M Glucose, 0.1 M citric acid, 80 mM KCl, 10 mM EDTA, adding fresh Triton X-100 to final 1% just before use). The nuclei were purified using 1x nuclei washing buffer (NWB, pH 5.0, 1 M Glucose, 0.1 M Na-citrate, adding fresh Triton X-100 to final 1% just before use). The purified nuclei were resuspended using 600 µl MNB buffer (50 % w/v Sucrose, 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 1 mM CaCl2) for MNase digestion at 37°C for 10 min. Centrifuge the digestion mix at 13,000 rpm for 10 min at 4°C and transferred supernatant into a new 1.5 ml tube. The digested nuclei pellet was resuspended in lysis buffer (1 mM Tris-HCl, pH 7.5, 0.1 mM PMSF, 2 % v/v Complete Mini) and leave it on ice for 1 h. After centrifugation, the supernatant was transferred into the 1.5 m tube containing digested chromatin. ChIP incubation buffer was added to the digested chromatin to make 1.7 ml total. The remaining steps were followed the published procedures (Zhang et al., 2012), including antibody incubation followed by adding protein A-sepharose beads, beads washing and ChIPed DNA elution, purification of ChIPed DNA for library preparation and sequencing on Illumina platform. The final purified DNA was used for ChIP-seq library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina purchased from NEB (Cat#: E7645S)