GSM8626473: Cre-neg-MFR; Mus musculus; RNA-Seq
Whole mesenteries and omenta from eight lipoatrophic AdiponectinCre/+ RosaDTAfl/+ female mice that received fat transplant surgery referred to as distal adipocyte receiving lipoatrophic mice (DARL) and their littermates that have standard fat depots that received sham surgery referred to as peritoneal adipocyte-replete control mice post-sham surgery (PARC) mice 8 weeks post-surgery were pinned on Sylgard dishes and kept hydrated in PBS. The adipose region in PARC mice and the corresponding area in DARL mice were carefully separated from the membranous sheet region for each omentum and mesentery using a scalpel and placed in RPMI with 5% FBS on ice. Each sheet or adipose region collected from each mouse was minced and digested separately using 1 mg/mL collagenase IV,  0.1 mg/mL DNAse, and 0.5% BSA for 30 minutes on a shaker. The samples were centrifuged and resuspended for downstream processing. Following digestion of the omentum and mesentery from PARC and DARL mice, the samples were washed and filtered with 100 m filters and stained for CD45+, DAPI, and TER-119. Samples were kept on ice until sorted on the FACSAria II with a 100-m nozzle (Becton Dickinson). Cells stained with DAPI were gated out during cell sorting to remove dead cells. Similarly, cells stained positive for TER-119 were excluded to remove red blood cells from our cell quantifications to increase the collection of desired cells. Approximately equal quantities of CD45+ and CD45- cells were submitted for 10X Genomics single-cell RNA sequencing via the McDonald Genome Institute Core. 10X Genomics Chromium Single Cell 5' Library and Gel Bead Kit