GSM8078774: Mouse neonate thymocytes, MRV infected; Mus musculus; RNA-Seq
The thymus was dissected with care to remove non-thymic tissue using scissors. The thymus tissue was placed into 1ml of 0.25% trypsin EDTA (GIBCO) at 37ºC. The tissue was incubated for 15 minutes. Dissociation and digestion were aided by vigorous pipetting using a large bore pipette every 5 minutes. Cells were filter through a 100μm filter into 9ml of RPMI 10% FBS to neutralize trypsin activity. Cells were then centrifuged at 1500rpm for 3.5 minutes followed by incubation in RBC lysis buffer (made in house) for 3 min at room temperature. Cells were then centrifuged again and washed twice in 9ml of PBS 0.5% bovine serum albumin (BSA). Cells were counted via hemocytometer then resuspended at 1200 cells/μL in PBS 0.04% BSA. The cells were kept on ice until ready for scRNAseq analysis. Single-cell 3' gene expression cDNA libraries were generated per manufacture protocols using the 10X Genomic Chromium Single-Cell 3' Library and Gel Bead Kit v1 and the 10x Chromium Controller