GSM8426156: Nsd1_E65ExE_Ctrl_H3K36me2_rep2; Mus musculus; ChIP-Seq
Embryos or cells were lysed in lysis buffer and subject to Mnase digestion, antibody immunoprecipitation, and subsequent library preparation. STAR ChIP-seq was performed as previously described (Zhang et al., 2016) with modifications. Briefly, samples were suspended and lysed followed by chromatin fragmentation by micrococcal nuclease (MNase, Sigma-Aldrich, N3755-200UN) at 37°C for 5 min. After being terminated, the supernatant containing chromatin was incubated with 1 μg of primary antibody (H3K36me2, Active motif, 61019; H3K36me3, Active motif, 61021 or 61902) at 4°C overnight with rotation. The next day, 100 μg of Protein A/G dynabeads (mixed at 1:1, Thermo Fisher Scientific, 10001D for Protein A and 10004D for Protein G) was added to each sample and incubated at 4°C for 3 hours with rotation. The beads were next washed five times with 150 μl of RIPA buffer and once with 150 μl of LiCl buffer. For each sample, beads were resuspended with 28 μl of H2O, 1 μl of 10× Ex Taq buffer (Takara, RR006B), and 1 μl of proteinase K (NEB, P8107S) and incubated at 55°C for 90 min, followed by incubation at 72°C for 40 min to inactivate the proteinase K. Samples were then subjected to TruSeq library preparation using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). Libraries were sequenced using the Illumina Hiseq X Ten or Novaseq 6000 system according to the manufacturer's protocol.