GSM8012228: lcmv,HTO, lung + spleen; Mus musculus; OTHER
Intestinal intraepithelial leukocytes were isolated as follows: small intestines were harvested and washed in PBS. Peyer's patches were surgically removed and the intestine was segmented in ~1 cm pieces prior to incubation with 1 mM dithiothreitol for 10 min at room temperature followed by addition of 30 mM EDTA and incubation for 30 min at 37oC. Intraepithelial cells were recovered from the supernatant of dithiothreitol and EDTA washes and mononuclear cells were isolated by collecting the middle ring after 40% and 80% gradient Percoll centrifugation. Immune cells from the lymph nodes, lungs, spleen, and liver were isolated by incubating the fragmented tissue in 1.5 ml HBSS supplemented with collagenase D at 400 U ml-1, 0.1 mg ml-1 Dnase 1 (Sigma) and 0.8 mg ml-1 dispase 1 (Sigma) for 30 min at 37oC. After digestion, tissue was forced 5 times through a 21-gauge needle and filtered through a 70 µm strainer into a 15 ml falcon tube with PBE. In addition to fluorescent antibodies, cells were co-stained prior to sorting with hashtag oligonucleotide (HTO)-labeled antibodies to CD45 and MHC-I for sample separation (two hashtags per sample) and HTO-anti-biotin for detection of the uLIPSTIC signal. Sorted cells were collected into a microfuge tube with 300 μl PBS supplemented with 0.4% BSA. After the sort, tubes were topped with PBS 0.4% BSA, centrifuged and the buffer was carefully reduced by removing the volume with a pipette to a final volume of 40 μl. Cells were counted for viability and immediately submitted to library preparation. The scRNA-seq library was prepared using the 10X Single Cell Chromium system, according to the manufacturer's instructions.