GSM1437740
GSM1437740: GRO-seq WT LiverZT16; Mus musculus; RNA-Seq
Mouse liver was harvested at indicated time and washed with cold swelling buffer (10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 2 U/ml Superase-In). Nuclear extract was prepared by dounce homogenization in cold swelling buffer and filtered using the cell strainer (100 µm, BD Biosciences). Lysate was centrifuged at 400g for 10 min, then resuspended in lysis buffer (swelling buffer with 10% glycerol and 1% Igepal) and incubated on ice for 5 min. Nuclei were washed twice with lysis buffer, and resuspended at the concentration of 10 x 10-7 nuclei/ ml in freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). The nuclear run-on assay was performed as previously described (Core et al., 2008; Step, 2014; Wang et al., 2011). Synthesis of cDNA from RNA was performed as described previously (Wang et al., 2011). cDNA was denatured at 70°C for 3 min then products from 105-400 nucleotides size were excised and eluted from shredded gel pieces for 4h in TE + 0.1% Tween and precipitated by adding 1 µl of glycogen, 300 mM NaCl and 2 volumes of ethanol and incubated overnight at -20°C. cDNA was resuspended into circularization reaction and circularized with CircLigase (Epicentre) for 1 hour at 60°C, denatured for 10 min at 80°C, and relinearized by adding relinearization mix (25 mM KCl, 500 µM DTT) and APE I (15U; New England Biolabs). Linearized DNA was amplified by PCR using Phusion Hot Start II Kit. The PCR product from 150-305 nucleotides size were eluted from shredded gel pieces and purified using ChIP DNA Clean and Concentrator Kit (Genesee Scientific).
GEO
GSM1437740
GEO Accession
GSM1437740