GSM3407994: H3K27me1_rep1; Arabidopsis thaliana; ChIP-Seq
Sperm nuclei from an ProHTR10:HTR10-Clover line were isolated by FACS and stored at -80°C in Cryopreservation Buffer (50 mM Tris-Cl pH 8.0, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 1 mM PMSF, 1x Roche protease inhibitor cocktail). Sperm mononucleosomes were generated by micrococcal nuclease (MNase, New England Biolabs) digestion. Aliquots of frozen sperm nuclei were thawed on ice and subsequently subjected to MNase digestion in MNase digestion buffer (1x NEB MNase reaction buffer, 1.5 mM DTT, 5% PEG 6000). EDTA was added to a final concentration of 10 mM to stop the MNase digestion. Nuclei were lysed by adding Triton X-100 and sodium deoxycholate to a final concentration of 0.1% each, incubated on ice for 15 minutes and then vortexed. Soluble material containing mononucleosomes were recovered after centrifugation and subjected to immunoprecipitation. Sequencing libraries were prepared using a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and sequenced on an Illumina Hiseq 2500 to generate 50 bp paired-end reads. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs)