GSM4820377: single_cell_RNAseq_epithelial_stemcells_gut_lane1_HTO_data; Mus musculus; RNA-Seq
Mice were euthanized with CO2, perfused via the left ventricle of the heart with 10 ml of PBS, siLP, liLP, lung and skin were collected and placed in cold complete medium (RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 20 mM HEPES, 50 mM b-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin). For siLP and liLP, Peyer's patches and mesenteric adipose tissue was removed. Tissues were open and wash in cold PBS to remove feces. Tissues were cut into 1-2 cm segments and treated with complete RPMI medium containing 5mM EDTA and 0.145 mg/ml dithiothreitol for 20 min at 37°C with constant stirring. Tissues were shake vigorously for 1 min, further digested with 10 ml 500 mg/ml DNase I (Sigma-Aldrich) and 100 mg/ml Liberase TL (Roche) with continuous stirring at 37°C. 2.5 x 10^4 of EpCAM+ intestinal epithelial cells were isolated by Sony MA900 cell sorter with from each sample. All samples were labelled with TotalSeqA hash tags (Biolegend), pooled, encapsulated into droplets using Chromium Single Cell Controller (10X Genomics) and libraries were prepared using Chromium Single Cell 3 Reagent Kits v3 (10X Genomics). Controller was overloaded with 2.5 x 10^4 cells per lane. The mRNA was prepared following the 10X Genomic user guide, while HTO library was prepared according to published guidelines