title: GSM7511050: single cell gene expression of immune cells from WT & IL33 KO BM12 heart grafts; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized and immediately perfused with PBS containing 0.5% heparin via the left ventricle. Heart tissue was minced by scissors and then placed in 10ml RPMI digestion buffer (10% FBS, 12.5mg collagenase IV (Thermo Fisher; 17104019) and DNAse I (Sigma Aldrich; DN25-1G)) and further homogenized using Miltenyi GentleMacs C tubes and Miltenyi tissue dissociator. The tissue homogenate was incubated in a shaker at 250 rpm and 37°C for 45 minutes. After digestion, homogenate was passed through a 70 μm strainers, centrifuged, and immune cells were then separated by a density gradient using Lympholyte-M (Cedarlane; CL5031) according to the manufacturer's protocol. Interphase cells were isolated, washed with RPMI, and then further purified using Miltenyi Biotec Debris removal solution (130-109-398) per manufacturer's instructions. The hashing protocol described by Stoekius et al in 2017 was used to generate a hastagoligo (HTO) library and a 3' gene expression library cell hashing and 3' gene expression