Multiplex Tissue Nuclei Profiling

Approximately 5 mg of tissue from each of the skeletal muscle, spleen, heart, liver, and fat samples was crushed into a fine powder in liquid nitrogen (Supplementary Table 1). Pulverized tissues were suspended in 1 mL ice-cold PBS and then gently spun down. The cell pellet was resuspended in 1 mL lysis buffer (50 mM HEPES with pH 7.5 [Life technologies, 15630-080], 140 mM NaCl [Ambion, AM9760G], 1 mM EDTA with pH 8.0 [Ambion, AM9260G], 10% glycerol [Sigma-Aldrich, G7757], 0.5% NP-40 [Roche, 11754599001], and 0.25% Triton X-100 [Amresco, 0694]), followed by isolation of 50,000 nuclei using a previously published protocol with minor modifications⁷⁹ . Then, the transposition reaction mix (12.5 μL TD buffer, 10 μL ddH₂O, and 2.5 μL TDE [Illumina, FC-121-1030]) was added to the isolated nuclei. The reaction system was incubated at 37 °C for 1 h followed immediately by purification with a QIAGEN MinElute PCR Purification Kit (QIAGEN, 28006). The transposed DNA fragments were amplified with the appropriate number of cycles as determined by qPCR. Size selection of the amplified PCR products (100–600 bp fragments) was performed by gel-purification informing previously studies^{79 –81 (link)}. Products from the size selection step were quantified and sequenced using an Illumina HiSeq X Ten PE150 platform.