Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Tissue (4-5 animals pooled per replicate) was homogenized 15x with a loose pestle in a glass homogenizer containing Homogenization Medium (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 1X Roche EDTA-free protease inhibitor cocktail, pH 7.8). 0.3% IGEPAL CA-630 was added, and the tissue was further dounced 5x with a tight pestle. After douncing, the homogenate was filtered through a 40 µm strainer.The nuclei were diluted 2:1 with cold, supplemented homogenization buffer, and 2mM EDTA was added. The sample was immediately sorted using the Sony SH800S Cell Sorter (purity mode) with a 100-μm sorting chip. 150,000 GFP+ nuclei were collected into CUT&RUN wash buffer. GFP- events were collected into CUT&RUN wash buffer, and 150,000 nuclei were subsequently counted on the Countess II FL Automated Cell Counter for ERα- and IgG negative control CUT&RUN. Takara SMARTer ThruPlex DNA-seq Kit