GSM9337357: Uninfected liver; Mus musculus; RNA-Seq
Mice were euthanized by CO2 asphyxiation and the liver was perfused with ice cold PBS by inserting a cannula through the portal vein. The liver was removed after perfusion, cut in small pieces and incubated in digestion solution (10% FCS, DNase1 1mg per 10ml, 10mg collagenase IV per 10ml) in a thermostatic bath at 37°C for 45min, stirring (100 rpm). Medium containing 10% FCS was then added to stop the enzymatic activity and the liver was smashed through 70 µm filter. The digested tissue was pelleted by centrifugation at 300g for 5min. Red blood cells (RBC) were lysed using RBC lysis buffer at room temperature for 5min. The pellets were suspended in 33% percoll and gently overlaid with phosphate buffered saline (PBS) and centrifuged at 300g for 20min without breaks. The cells were collected from the bottom of the tube. Liver cell suspensions isolated from 5 uninfected and 5 S. aureus-infected mice (24 h of infection) were pooled and loaded onto the 10x Chromium Single Cell instrument (10x Genomics). Libraries were prepared from single-cell suspensions using sc 3'GEM-X v4 1-plex 20k according to the 10x Genomics protocol and sequenced using an Illumina NovaSeq X 1,5B 300 cycle sequencer (Illumina) with a sequencing depth of 200 million reads per sample