GSM2087173: iWAT, GRO-seq, Rev-erb alpha KO; Mus musculus; OTHER
The nuclear run-on assay was performed as previously described (Core et al., 2008; Step et al., 2014; Wang et al., 2011). Nuclei extraction: Mouse tissues were harvested at Zeitgeber Time 10 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 in freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). 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. Synthesis of cDNA from RNA was performed as described previously (Wang et al., 2011) using oNTI223 primer (5′-/5Phos/GA TCG TCG GAC TGT AGA ACT CT/idSp/CAA GCA GAA GAC GGC ATA CGA TTT TTT TTT TTT TTT TTT TTV N-3′) where the “5Phos” indicates 5′ phosphorylation, “idSp” indicates the abasic dSpacer furan, and “VN” indicates degenerate nucleotides. The reaction was treated with exonuclease I (Fermentas) for 15 min at 37°C, followed by 100 mM NaOH for 20 min at 98°C, and neutralized with 100 mM HCl. cDNA was denatured at 70°C for 3 min, and run on 10% TBE-urea gel, 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, according to manufacturer’s instructions. The oligonucleotide primers oNTI200 (5′-CAA GCA GAA GAC GGC ATA-3′) and oNTI201 (5′-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAC G-3′) were used for amplification. The PCR product was run on a 10% TBE gel and products from 150-305 nucleotides size were eluted from shredded gel pieces for 4h in TE + 0.1% Tween + 150 mM NaCl and purified using ChIP DNA Clean and Concentrator Kit (Genesee Scientific). Libraries were sequenced on an Illumina HiSeq2000 with sequencing primer 5′-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3′.
GEO Accession
GSM2087173