GSM9231497: aging adipose tissue, multiplexed library; Mus musculus; RNA-Seq
Nuclei were isolated from frozen adipose tissue samples using a modified protocol adapted from a previously described method (5). For each sample, approximately 50 mg of frozen adipose tissue was placed into a gentleMACS C tube (Miltenyi Biotec) containing 2 ml of freshly prepared TST buffer (10 mM Tris-HCl, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 0.01% BSA, 0.03% Tween 20, and 0.2 unit/μL RNasin Plus). Tissues were dissociated using a gentleMACS Dissociator (Miltenyi Biotec) with the program “mr_adipose_01” performed twice, including a 5-minute incubation on ice between each run. After an additional 10-minute incubation on ice, the lysate was passed through a 40 µm nylon filter and collected into a 50 mL conical tube on ice. The filter was rinsed with 3 mL of freshly prepared ST buffer (10 mM Tris-HCl, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2 and 0.2 unit/μL RNasin Plus). Sample were then centrifuged at 500 g for 5 minutes at 4 °C with the brake set to low. The nuclear pellet was washed three times by resuspending in resuspension buffer (1% BSA and 0.2 unit/μL RNasin Plus in PBS) followed by centrifugation. A 10 μL aliquot was used to determine nuclei quality and count using NucleoCounter NC-3000 (ChemoTetec) after staining with Solution 13 (AO•DAPI, ChemoTetec). Nuclei were fixed according to the low volume fixation protocol (ScaleBio). Fixed samples were thawed on ice, recounted, and adjusted to 2,000 nuclei/ml with ScaleBio wash buffer (Part No. 202100001) prior to loading. Each sample was loaded into 2 wells of the initial RT 96-well plate with 10,000 nuclei per well. The plate was incubated, and nuclei in the wells were pooled to re-distribute into the second 384-well ligation plate. Following ligation, nuclei were pooled again and counted to distribute into the final distribution plates (96-well plates) with 1,600 nuclei per well. The library was prepared and pooled from the final distribution plates with a target recovery of 100,000 nuclei per final distribution plate.