GSM8408110: brown adipocyte, ATAC-seq, repeat 1; Mus musculus; ATAC-seq
SVF cells were obtained from adipose tissue in the brown fat of male C57BL/6J mice at 3-4 weeks old. Type I collagenase (2mg/ml, Sigma, SCR103) was preconfigured using DMEM basic medium (Gibco, C11995500BT) containing 2% volume of FBS (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, and stored in the dark after removal of bacteria by filtration using a 0.22 μM filter. The volume ratio of collagenase to adipose tissue was maintained at 2:1. Then the adipose pad was cut into pieces to 1mm³ and digested in 37℃ water bath for 1h, during which the tube was reversed and mixed every five minutes. The digested adipocytes were filtered (70 μm), then centrifuged at 1000 rpm for 10 min. After re-suspension with PBS, centrifuge at the same speed and time. DMEM/F-12 (Meilunbio, MA0214) containing 10% FBS (Gibco) and 1% penicillin-streptomycin was used to culture the primary SVF, and then the cells were implanted into the pore plate of appropriate size. The medium was changed 24 h later, and then every 48 h until the cells grew to contact inhibition state. The plate-fully-covered cells then subjected to differentiation. As for brown adipogenesis, culture medium was added insulin (5μg/ml, sigma, I3536), Dexamethasone (5μM, sigma, D4902), 3,3',5-Triiodo-L-thyronine (T3, 1nM, sigma, T2977), Rosiglitazone (1μM, sigma, R2408), Isobutyl methylxanthine (0.5mM, sigma, I5879). The above differentiation medium were changed 48 h later with medium only containing insulin, T3 and rosiglitazone, and renewed every 48 h. Cells were incubated at 37℃ in a suitable atmosphere containing 5% CO2. Native nuclei were purified from adipocytes，and washed twice gently with wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail). For ATAC seq, the Nextera DNA Library Preparation Kit (Illumina) was used to perform the transposition according to the manufacturer's manual. 50,000 nuclei were pelleted and resuspended with transposase, for 30 minutes at 37 ℃. The transposed DNA fragments were purified immediately after with a MinElute PCR Purification Kit (Qiagen). After Samples were PCR-amplified using 1X NEBNext High-Fidelity PCR Master Mix (New England Biolabs, MA). Subsequent libraries were purified with the MinElute PCR Purification Kit (Qiagen). ATAC Libraries were pooled to a final concentration of 10nM and sequenced paired-end using the Illumina NovaSeq 6000.