title: GSM7869034: Cerebrum, input, H3K27me3; Mus musculus; ChIP-Seq
library_construction_protocol: Nuclei were isolated as previously described (Krishnaswami et al, 2016). Briefly, tissue was chopped, flash frozen and stored at -80 ºC. The Dounce homogenizer (2 ml one), pestles and all the buffers (NIM1 – 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris pH 8; NIM2 – NIM1, 1 µM DTT, protease inhibitor; Homogenization buffer – NIM2, 0,4 U/µl RiboLock (ThermoFisher, EO0384)) were precooled. The tissue was homogenized in 1 ml homogenization buffer with five strokes of the loose pestle, followed by 15 strokes of the tight pestle. Homogenate was filtered through a BD Falcon tube with a 35-μm nylon cell strainer cap (352235, Corning). The number of nuclei was counted by staining an aliquot with trypan blue and using TC10 Automated Cell Counter (BioRad). The nuclei were resuspended in 1 ml NIM2 buffer and sample was centrifugated (500 g, 5 min, 4 ºC). The washing step was repeated 2-3 times. Further ULI-ChIP-seq protocol was followed (Brind'Amour, 2015). Briefly, around 1 million nuclei per IP were diluted in 50 µl Nuclear storage buffer (15% sucrose, 10 mM Tris pH 7,2, 70 mM KCl, 2 mM MgCl2 and proteinase inhibitors) and fragmented using MNase at 37 ºC for 7,5 min. Upon fragmentation reaction was stopped with 6,6 µl 100 mM EDTA and 6,6 µl 1% Triton/1% DOC. The samples were then diluted up to 200 µl with Complete immunoprecipitation buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 15 mM NaCl, 0.1% Triton X-100, 1x EDTA-free protease inhibitor cocktail, 1mM PMSF). The eluted DNA was purified by phenol–chloroform, precipitated with EtOH, and resuspended in 20 µl MQ. For library preparation the Kapa Hyper Prep Kit (Roche, KK8504, 07962363001) was used, following the manufacturer's instructions.