GSM7814535: HDAC3_Control_7d_Rec; Mus musculus; ATAC-seq
Interscapular BAT was harvested from individual mice and snap frozen in liquid nitrogen. For each genotype and condition, three biological replicates (individual mouse BAT pads) were thawed on ice and quickly pooled in ice-cold celling buffer A and nuclei isolated on ice by dounce homogenization per GRO-seq nuclei isolation protocol. 5 x 104 isolated nuclei were then washed in ice-cold 1ml of 1x Tagmentation Buffer (2x 20 mM Tris (hydroxymethyl)-aminomethane, 10mM MgCl2, 20% (vol/vol) dimethylformamide, pH 7.6) and resuspended in 50 ml 1x TD buffer prior to the transposition assay. The Nextera DNA Library Prep Kit (Illumina, FC-121-1030) was used for the transposition reaction. The nuclei pellet was resuspended and incubated with transposition reaction mix at 37°C for 30 minutes. DNA was isolated using MinElute Reaction Cleanup kit and eluted in elution buffer (EB). Libraries were generated using the Ad1_noMX and Ad2.1–2.4 barcoded primers and were amplified for 7-9 total cycles. The libraries were purified with AMPure beads (to remove primer-dimers and >1,000 bp DNA fragments) by manufacturer protocol. The library was assessed on an Agilent High Sensitivity DNA Bioanalysis chip and quantitated with the Qubit dsDNA high-sensitivity Assay kit (Thermo Fisher Scientific, Q32851). Libraries were sequenced on an Illumina HiSeq 2000 using 50 bp paired-end (50PE) sequencing.