GSM6858788: Adipocyte, Flox, H3k27ac ChIP; Mus musculus; ChIP-Seq
Mature adipocytes were obtained from adipose tissue in the inguinal position of male mice at 8-10 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. Up layer mature 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; H3K27ac, Active Motif, #91193, 1:50; normal rabbit IgG, Merck Millipore #12-370; normal mouse IgG, Merck Millipore #12-371) and second antibodies (anti-rabbit IgG antibody, goat monoclonal: Millipore AP132; anti-mouse IgG antibody, goat polyclonal: Millipore AP124). 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). 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). All CUT&Tag and ATAC Libraries were pooled to a final concentration of 10nM and sequenced paired-end using the Illumina NovaSeq 6000.