title: Single cell RNA sequencing of adipose tissue in germ-free and specific pathogen free mice
design: Single cell RNA sequencing of adipose tissue in germ-free and specific pathogen free mice
library_construction_protocol: Epididymal-adipose tissue from mice was dissected and placed in 5 ml of DMEM (without phenol red, 4.5 g/l glucose) at room temperature and disintegrated by chopping with sterile scissors. Subsequently, 0.2 g/ml of Collagenase II was added, and samples were incubated for 20 min at 37 °C while shaking. Subsequently, 5 ml of cold DMEM was added to each sample and filtered through a 250-μm mesh. Samples were centrifuged at room temperature for 5 min at 300g without breaks and with slow acceleration. The pellets containing immune cells as well as top layer containing adipocytes were recovered using glass Pasteur pipettes. Immune cell fraction was resuspended in 0.5 ml ACK buffer (Gibco, A1049201), and incubated for 1 min at room temperature. Then, 14 ml of PBS was added, and cells were collected by centrifugation at 500g for 10 min. Pellets were resuspended in Ca/Mg-free PBS with 0.04% BSA, filtered through a 40-µm mesh. To adipocyte fraction was three times the volume (i.e. ~3 ml) of ice-cold NST Lysis Buffer (0.1% NP40, 10 mM Tris-HCl, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 40 U/ml RNase Inhibitor)23 was added and incubated on ice for 5 min to isolate nuclei, then sample was centrifugated for 5 min at 500g at 4 °C and resuspended in 1ml of Wash Buffer (Ca/Mg-free PBS, 1% BSA, 0.2U/μl RNase Inhibitor). Wash was repeated, nuclei were filtered through 65-μm mesh and counted using Trypan blue. Fractions were combined to obtain 50% cell fraction and 50% nuclei fraction before loading onto 10x Genomics Chromium. according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide