GSM5708090: Liver_AktDKO_refed_2h; Mus musculus; OTHER
Nuclear Run-On (NRO): Nuclei were mixed with an equal volume of run-on buffer (10 mM Tris pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 200 U/ml Superase-In, 1% Sarkosyl, 500 μM ATP, GTP and Br-UTP, 2 μM CTP) and incubated for 5 min at 30°C. Nuclear RNA was extracted with Trizol (Invitrogen) and treated with DNase (Ambion). RNA was hydrolyzed using fragmentation reagents (Ambion) for 13 min at 70°C and purified through a Micro Bio-Spin Column P-30 (Bio-Rad). RNA was treated with T4 polynucleotide kinase (New England Biolabs) for 1h at 37°C, then with an additional T4 polynucleotide kinase for 1h more, and inactivated for 5 min at 75°C. NRO-RNA immuno-precipitation: Anti-BrdU agarose beads (Santa Cruz) were rotated for 1h in blocking buffer (0.25x SSPE, 38 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.1% PVP, and 0.1% BSA). Then NRO-RNA was rotated with beads for 1h, followed by washes twice in binding buffer (0.25x SSPE, 38 mM NaCl, 1 mM EDTA, 0.05% Tween-20), twice in low salt buffer (0.2x SSPE, 1 mM EDTA, 0.05% Tween-20), once in high salt buffer (0.2x SSPE, 1 mM EDTA, 0.05% Tween-20, 138 mM NaCl), and twice in TET buffer (TE pH 7.4, 0.05% Tween-20). BrdU-labeled RNA was eluted from the beads four times for 10 min with 100 μl elution buffer pre-heated to 42°C. RNA was precipitated by adding 300 mM NaCl, 1 µl of glycogen and 2.5 volumes of ethanol and incubated overnight at -20°C. RNA was resuspended in water (with 1 U/µl Superase-In and 0.05% Tween-20), denatured for 3 min at 65°C, and treated with poly(A)-polymerase (New England Biolabs) for 30 min at 37°C. Library construction 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).