GSM3209269: GmLM_H3K27me3_br2; Glycine max; ChIP-Seq
Late maturation embryo nuclei were isolated on a Percoll/sucrose gradient and the nuclei pellet was frozen prior to sonication. Chromatin isolation and immunoprecipitation were performed as described in Gendrel AV, Lippman Z, Martienssen R, Colot V. Nat Methods. 2005;2:213–218, except that the isolated nuclei were resuspended in lysis buffer described in by Johnson et al. (2002) and sonicated to achieve chromatin fragments ranging from 100 to 500 bp.  Nuclear extracts  were incubated overnight with either 3ug of anti-Histone H3 (Abcam, ab1791), 1ug of anti-H3K4me3 (Abcam, ab8580), or 10ug of anti-H3K27me3 (Abcam, ab6002).  Crosslinks were reversed and protein digested as described in Dahl and Collas, Nat Protoc. 2008;3(6):1032-45, extracted with phenol:chloroform and precipitated using standard procedures.  Late maturation embryo nuclei were isolated on a Percoll/sucrose gradient and the nuclei pellet was frozen prior to sonication. ChIP libraries were generated using Nugen Ovation Ultralow DR Multiplex System, following the manufaturer's instructions and enriched by PCR using 15 cycles.  Libraries were size-selected by electrophoresis and purified using Qiagen MinElute gel purification kit