GSM8796393: Mammary glands, Oxtr flox rep (1-4); Adipo-specific-Oxtr KO rep(1-4), HTO; Mus musculus; RNA-Seq
Nuclei were isolated from frozen mouse mammary gland samples for 10x snRNA-seq using a slightly modified approach to what was previously described (PMID: 32888429 and PMID: 35296864). Samples were kept frozen on dry ice until immediately before nuclei isolation, and all sample handling steps were performed on ice. Each flash-frozen adipose tissue sample was placed into a gentleMACS C tube (Miltenyi Biotec, 130-096-334) with 2 ml freshly prepared TST buffer (0.03% Tween 20 (Bio-Rad 1610781), 0.01% Molecular Grade BSA (New England Biolabs B9000S), 146 mM NaCl (ThermoFisher Scientific AM9759), 1 mM CaCl2 (VWR 97062-820), 21 mM MgCl2 (Sigma Aldrich M1028), and 10 mM Tris-Hcl pH 7.5 (ThermoFisher Scientific 15567027) in Ultrapure water (ThermoFisher Scientific 10977-023)) with or without 0.2 U µl−1 of Protector RNase Inhibitor (Sigma Aldrich 3335399001). gentleMACS C tubes were then placed on the gentleMACS Dissociator (Miltenyi Biotec 130-093-235) and tissue was dissociated by running the program mr_adipose_01 twice, and then incubated on ice for 10 min. Lysate was passed through a 40 µm nylon filter (CellTreat 229481) and collected into a 50 ml conical tube (Thomas Scientific 1149Z03). Filter was rinsed with 3 ml of freshly prepared ST buffer buffer (146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2; 10 mM Tris-Hcl pH 7.5) with or without 0.2 U µl−1 RNase Inhibitor, and collected into the same tube. Flow-through was centrifuged at 500g for 5 min at 4 °C with brake set to low. Following centrifugation, supernatant was removed, and the nuclear pellet was resuspended in 50–200 µl PBS pH 7.4 (ThermoFisher Scientific 10010049) with 0.02% BSA, with or without 0.2 U µl−1 RNase inhibitor. In order to reduce ambient mRNA, the nuclear pellets of some samples were washed 1–3 times with 5 ml PBS, 0.02% BSA before final resuspension. An aliquot of nuclei from each sample was stained with NucBlue (Invitrogen R37605) counted in a haemocytometer using fluorescence to identify intact nuclei, and then immediately loaded on the 10x Chromium controller (10x Genomics) according to the manufacturer's protocol. For each sample, 10,000–16,500 nuclei were loaded in one channel of a Chromium Chip (10x Genomics). The Single Cell 3′ v3.1 chemistry was used to process all samples. cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). cDNA and gene expression library fragment sizes were assessed with a DNA High Sensitivity Bioanalyzer Chip (Agilent 5067-4626). cDNA and gene expression libraries were quantified using the Qubit dsDNA High Sensitivity assay kit (ThermoFisher Scientific Q32851). Gene expression libraries were multiplexed and sequenced on the Nextseq 500 (Illumina) with a 75-cycle kit and the following read structure: Read 1: 28 cycles, Read 2: 55 cycles, Index Read 1: 8 cycles.