GSM7814545: BAT_WT_22C; Mus musculus; OTHER
Ten genotype confirmed mice per group per experimental condition were utilized for each experiment. Ten interscapular BAT pads from each HDAC3 KO and control littermate groups were quickly dissected, pooled and then minced in ice-cold nuclei isolation buffer A (10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 2 U/ml Superase-In), a cold swelling buffer, and filtered through a 100 um cell strainer. All GRO-seq steps including tissue harvest, nuclei isolation, nuclear run-on reactions, immunoprecipitations, library preparation, and sequencing were performed in parallel to reduce batch effects. Nuclei Isolation: Nuclei were isolated by dounce homogenization conducted on ice. Filtered lysate in buffer A was centrifuged at 400 x g for 10 min and resuspended in lysis buffer (swelling buffer with 10% glycerol and 1% Igepal). Lysis buffer containing sample were incubated on ice for 5 min, and nuclei washed twice with lysis buffer. Nuclei pellet was washed twice with lysis buffer. Nuclei were resuspended at a final concentration of 10 x 107 nuclei/ml in freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA).  Nuclear Run-on reaction: 40 million nuclei were utilized for each nuclear run-on reaction. The nuclei were thawed on ice and mixed with 1:1 volume of ice-cold 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 uM ATP, GTP and Br-UTP, 2 uM CTP). The run-on reaction was incubated at 30C for 7 minutes and terminated by addition of Trizol with 5 minute incubation at room temperature. RNA was extracted from Trizol, treated with DNAse, prior to 10 minute RNA hydrolysis fragmentation reaction, purified over a Micro Bio-Spin P30 column and treated with T4 polynucleotide kinase at 37C for 1 hour, treated with additional T4 polynucleotide kinase and heat inactivated for 5 minutes at 75C.  NRO-RNA Immunoprecipitation: Blocked Anti-BrdU agarose beads (Santa Cruz Biotechnology, sc-32323 AC), 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) were used for immunoprecipitation. Labeled-RNA was incubated and rotated with beads for 1h at room temperature, followed by washing beads 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 anti-BrdU agarose beads four separate times using 10 min incubation with 100 ul preheated (42C) elution buffer. RNA precipitation was performed using 300 mM NaCl, 1 ul of glycogen and 2.5 volumes of ethanol and an overnight incubation at −20C, followed by 30 minute centrifugation at 4C. RNA pellet was resuspended in water (with 1 U/μl Superase-In and 0.05% Tween-20), denatured for 3 min at 65C, placed on ice, and subsequently treated with E.coli poly(A)-polymerase for 30 min at 37C. cDNA synthesis was completed utilizing entire volume of E.coli poly(A)-polymerase reaction with previously reported oNTI223 primer (5′-/5′ phosphorylation/GA TCG TCG GAC TGT AGA ACT CT/abasic dSpacer furan/CAA GCA GAA GAC GGC ATA CGA TTT TTT TTT TTT TTT TTT TT/ degenerate nucleotides /-3′) using Superscript III RT (Thermo Fisher Scientific, 18080-051) in reaction mix for 40 min at 48C. cDNA reaction was incubated with exonuclease I for 15 min at 37C, then inactivated using 100 mM NaOH for 20 min at 98C, and neutralized with 100 mM HCl. Denatured cDNA was run on a 10% TBE-urea 10-well gel, gel stained with SYBR-gold, and gel extracted 105-400 nucleotides size were excised, eluted from shredded gel pieces in TE + 0.1% Tween for 4 hours at room temperature and then ethanol precipitated. cDNA was resuspended and circularized with CircLigase for 1 hr at 60C, denatured for 10 min at 80C, and relinearized with relinearization mix (25 mM KCl, 500 μM DTT) plus 15 unites APE I enzyme. PCR amplification was conducted with Phusion Hot Start II Kit (Thermo Fisher Scientific, F-549L), according to manufacturer's instructions. PCR amplification was performed using 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′). PCR amplified product was run on a 10% TBE gel and products ranging from 150-305 nucleotides were gel extracted and eluted from gel pieces using 4 h in TE + 0.1% Tween + 150 mM NaCl. Gel isolated PCR products in elution buffer were purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research, 11-379). Libraries were sequenced on an Illumina HiSeq2000 with sequencing primer 5′-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3′.