GSM6288583: Replicate 3 from 21 days post infection, scRNAseq; Mus musculus; RNA-Seq
GSM6288583
Mouse livers were cut into small pieces with a scalpel and crushed against a 50mm stainless steel mesh plate.  The mesh plate was rinsed with cold MACS buffer (PBS with 0.6% FBS and 0.5M EDTA) to release the cells into the collection plate.  Cells were washed with MACS buffer and centrifuged at 50G for 3 minutes at 4°C to get rid of most of the hepatocytes and the supernatant was collected (non-parenchymal cells).  The non-parenchymal cells were then centrifuged at 300G for 7 minutes at 4°C.  The pellet was resuspended in 10mL of 40% Percoll and overlaid with 70% Percoll.  The cells were centrifuged at 800G for 20 minutes at 4°C with no brake and the interphase with cells were collected and resuspended up to 15mL in MACS buffer.  The cells were centrifuged at 300G for 7 minutes at 4°C and the cell pellet was resuspended then counted with the BioRad cell counter (Bio-Rad, TC20). 0.25-0.5 million non-parenchymal cells were used with the EasySepTM Mouse T cell Isolation Kit (Stemcell Technologies, Cat. #19851) for negative selection of T cells, following kit protocol.  Briefly, cells are stained with a cocktail of antibodies bound to magnetic particles and specific for non-T cells. These cells are retained against the wall of the FACS tube when the EasySep magnet is applied, and the remaining cells (T cells) are poured out of the tube and counted for sequencing. Library was performed according to the manufacter's instructions (10x Genomics scSeq VDJ Reagents). scRNA-seq (5' Gene Expression library) with V(D)Jseq (TCR)