GSM6284722: Cold_PBZ_3; Mus musculus; ATAC-seq
Briefly, for nuclei preparation, BAT pads were homogenized (with pestle A) in a pre-chilled 2 ml Dounce homogenizer containing 2 ml cold 1x Homogenization Buffer (60 mM Tris pH 7.8, 30 mM CaCl2, 18 mM MgAc2, 320 mM sucrose, 0.1 mM EDTA, 0.1% NP40, 0.1mM PMSF, 1mM b-mercaptoethanol). The resulting solution was pre-cleared using a 100 μm filter and grounded 20 times (with pestle B). To generate the iodixanol gradient, 1 volume (800 μl) of 50% Iodixanol solution was added to 800 μl of grounded BAT solution to give a final concentration of 25% Iodixanol in a 5 ml Low-Bind Eppendorf tube. Then, 1.2 ml of 29% Iodixanol solution was added under the 25% mixture, and another 1.2 ml of 35% Iodixanol solution wad added under the 29% mixture. The gradient was centrifuged 3,000 g for 20 minutes at 4°C with the brake off. The nuclei band was collected into a new tube and nuclei were counted using trypan blue staining and Countess® II FL automated cell counter. 50,000 nuclei were transferred into a tube containing 1 ml of ATAC-RSB+0.1% Tween-20 and centrifuged 500 g for 10 minutes at 4°C. For optimized transposition, Omni-ATAC ATAC-seq reaction mix (25 μl 2x TD buffer, 100 nM transposase, 16.5 μl PBS, 0.01% digitonin, 0.1% Tween-20)58 was added to the nuclei pellet. Nuclei were resuspended by pipetting up and down 6 times. The resulting solution was incubated at 37°C for 30 minutes in a thermomixer (1,000 RPM). For the pre-amplification of transposed fragments, solution was cleaned with a Zymo DNA Clean and Concentrator-5 kit  (Cat. No. D4014) according to the manufacturer's instructions. DNA was eluted in a 21 μl elution buffer and amplified for 5 cycles using NEBNext 2x MasterMix (NEB, M0541L) as previously described (PMID: 28846090), using published primers (PMID: 24097267). 10% of the pre-amplified mixture, was used to run a qPCR to determine the number of additional cycles needed as previously described58. Next, the amplification profiles were manually assessed and the required number of additional cycles were determined as previously described59. The final PCR reaction was purified using a Qiagen MinElute PCR Purification Kit and eluted in 20 μl elution buffer. A subsample of each library was diluted to 1:1,000 to fall within range of the standards to perform concentration quantification using the KAPA Library Quantification Kit (Cat No. KK4854) according to the manufacturer's instructions.