GSM7928121: Primary beige adipocyte,  Ets1 ChIP; Mus musculus; ChIP-Seq
SVF cells were obtained from adipose tissue in the inguinal position 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 beige 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. 6 days later, the mature beige adipocyte were collected and treated according to the manufacturer's instructions by using in situ DNA Binding Library for Illumina Cut&Tag kits (Yeasen, 12598E12). 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 CUT&Tag, 1x10^5 nuclei bound to Concanavalin A-coated beads were incubated with indicated primary antibodies (Ets1, Active Motif, #39581, 1:50) and second antibodies (anti-rabbit IgG antibody, goat monoclonal: Millipore AP132). Transposase pA-Tn5 was used to induce DNA tagmentation, followed by Proteinase K digestion and PCI extraction. The ethanol precipitated DNA was immediately subjected to PCR amplification by Universal and barcoded i7 primer using NEBNext HiFi 2x PCR Master mix. The amplified library was purified without size selection. ChIP-seq libraries were prepared from 3-5ng DNA using NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S), according to the manufacturer's instructions. Post-amplification libraries were size selected at 250–450bp in length using AMPure XP beads ((Beckman Counter)) and were quantified using the Library Quantification Kit for Illiumina (Kapa Biosystems, KK4603). The CUT&Tag Library was pooled to a final concentration of 10nM and sequenced paired-end using the Illumina NovaSeq 6000.