GSM6533609: Fasted4 (ATAC); Mus musculus; ATAC-seq
Approximately 50 -100 mg of fresh tissue was homogenized via dounce homogenizer in 1 ml of nuclear isolation buffer (20 mM Tris-HCl, 50 mM EDTA, 5 mM Spermidine, 0.15 mM Spermine, 0.1% mercaptoethanol, 40% glycerol, 1mM EGTA, 60 mM KCl, 1% IGEPAL pH 7.5) and filtered through a 40 M nylon filter. Samples were centrifuged at 4 °C at 1000 × g for 10 min and the pellet was resuspended with 1 ml cold resuspension buffer (RSB) (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH 7.4). Approximately 50,000 nuclei from these samples were removed and centrifuged at 4 °C at 500 × g for 5 min. Supernatant was removed and the transposase reaction was performed immediately as described in Buenrosto et al., 2015. For ATAC-Seq, size selection was done with AMPure XP magnetic beads. Libraries were quantified by qPCR using NEBNext Library Quant Kit for Illumina