GSM6912031: P28_12wks_F_PROseq; Homo sapiens; OTHER
Frozen tissue was pulverized using a liquid nitrogen-chilled hammer mill, homogenized in ice-cold Swelling Buffer (10 mM Tris-HCl pH 8, 2 mM magnesium acetate, 3 mM calcium chloride, 0.25mM DTT, and 1x cOmplete protease inhibitor cocktail (Roche, 11697498001)), and passed through a 40 micron filter.  The nuclei were released from the pulverized tissue in Lysis Buffer   (Swelling Buffer containing 10 mM NaCl, 300 mM sucrose, and 0.5% NP-40), washed three times in Lysis Buffer  , and flash frozen in Nuclei Freezing Buffer (50 mM Tris-HCl pH 8.3, 5 mM magnesium chloride, 0.1 mM EDTA, 40% glycerol) in aliquots of 5 x 106 nuclei. Nuclear run-on was performed at 37°C for 5 minutes using biotin-labeled CTP.  PRO-seq libraries were constructed following the protocol of Mahat and colleagues (Mahat et al., 2016)