title: GSM8251148: WT, replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: To isolate liver inflammatory cells, the liver was excised, minced into small pieces, and placed in a tube containing cold DMEM (4.5 g/L glucose) 10% FBS, 0.75 mg/mL collagenase A and 50 ul/mL DNaseI. Then, the solution containing liver pieces was mixed at 37ºC for 30 min at 2000 rpm to digest. On top of a 50 mL falcon, liver tissue was added to nylon mesh strainer and 2-3 ml DMEM media (4.5 g/L glucose) was added. Liver tissue was mashed through the mesh with the plunger from a 3 ml syringe, until volume was brought up to 15 ml with cold DMEM. After centrifugation at 500g for 5 min at 4°C, pellet was resuspended in 1 mL of RBC lysing buffer and incubated 5 min at RT. Reaction was stopped by adding 10 mL of DMEM (4.5 g/L glucose) supplemented with 10% FBS. After centrifugation at 500g for 5 min at 4 °C, supernatant was discarded, and the pellet was resuspended in 15 mL of 38% Percoll in DMEM media (4.5 g/L glucose) supplemented with 10% FBS and underlaid with 70% Percoll in PBS and further centrifuged at 500g for 30 min at 4°C. Interface with mononuclear cells was collected and washed with 15 mL of DMEM media (4.5 g/L glucose) supplemented with 10% FBS. After centrifugation at 500g for 10 min at 4°C, supernatant was discarded and the pellet was washed and finally resuspended in 5% BSA, 2 mM EDTA in Ca2+/Mg2+ -free PBS (MACS buffer). CD45+ cells were isolated using FACSARIA (30M). 5 mice were pooled to obtain the required number of cells. Fixed cell suspensions obtained with the "Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit" (10xGenomics PN-1000414) were processed with the "Chromium Fixed RNA Profiling Reagent Kit" according to the manufacturer's instructions (10xGenomics, user guide CG000527). Briefly, individual samples were hybridised to *mouse* transcriptome probe sets (Chromium Fixed RNA Kit, 10xGenomics *PN1000496*), multiplexed and encapsulated in gel beads in emulsion (GEMs) in a Chromium iX instrument (10xGenomics). GEM recovery and gene expression library construction were all performed as recommended by the manufacturer. Libraries were sequenced on an Illumina instrument ((Illumina NovaSeq X) by following manufacturer's protocols.