GSM2221748: ChIP-seq, BAT H3K27ac, Adiponectin-Cre HDAC3 floxed, 29C/5pm, replicate 2; Mus musculus; ChIP-Seq
ChIP-seq: Interscapular BAT was isolated and processed separately for each sacrificed mouse. The tissues were immediately minced, crosslinked in 1 % formaldehyde/PBS, and stored at -80 C. For each fat pad, material from 3-4 mice was used for sequencing. To prepare ChIP extracts, frozen tissue was thawed, immediately crosslinked, and suspended ChIP buffer (50 mM HEPES/NaOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate and Complete protease inhibitor) supplemented with 0.1% SDS. Samples were incubated at 4 C for 10 min, and subjected to microtip probe sonication under conditions optimized for IP efficiency. Extracts were cleared of insoluble lipid by microfuge centrifugation at full speed followed by extract transfer to a new tube. The process was repeated until minimal lipid accumulated at the top of the extract after centrifugation. GRO-seq: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). ChIP-seq: ChIP-Seq libraries were produced and sequenced according to Illumina protocols. Libraries were multiplexed for sequencing.GRO-seq: 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'.