title: GSM6721498: subcutaneous white adipose resident eosinophils m2771; Mus musculus; RNA-Seq
library_construction_protocol: Peripheral blood was collected by cardiac puncture, subjected to red blood cell lysis. Adipose tissue was digested at 37C for 45 min with 1 mg/mL collagenase type II to isolate the stromal vascular fraction (SVF). The SVF was pelleted by centrifugation and then subjected to red blood cell lysis. Blood and adipose SVF cells were pre-blocked with Fc Block then stained with CD45-biotin/streptavidin-BV711, CD11b-FITC, F4/80-PECy7, and Siglec-F-BV421, prior to FACS sorting. TOPRO3 was used as a viability dye. Eosinophils were sorted using a BD FACS Aria III and defined as CD45+ CD11b+ SiglecF+ live cells. Cells were sorted into FACS buffer containing SUPERase-In RNase Inhibitor then carried forward for RNA isolation. RNA was extracted from sorted eosinophils using the Single Cell RNA Purification Kit (Norgen). RNA quality was assessed using a Bioanalyzer Pico chip (Agilent) and cDNA libraries prepared using SMARTer Stranded Total RNA-Seq Kit v2 Pico Input Mammalian (ClonTech/Takara Bio).