title: GSM8072564: Experiment_1_cDNA; Mus musculus; RNA-Seq
library_construction_protocol: Livers were cut to small pieces in 5 ml liver digest medium, squashed, and incubated at 37⁰c for 30 min. PBS was added and cells were centrifuged at 350g for 5 min at 4⁰c. Further digestion was done with Accumax+DNAse for 10 min at 37⁰c followed by neutralization with FBS and PBS. Suspension was filtered through a 70 µm mesh. Two centrifugations at 30g for 5 min at 4⁰c were performed to reduce hepatocytes content. Supernatant was then centrifuged at 350g at 4⁰c to precipitate the immune cells, and the pellet was resuspended in 6ml of 35% Percoll and gently placed on top of 3ml of 75% Percoll. Cells were centrifuged for 20 min at 900g without brake, and the ring of immune cells between the 2 Percoll concentrations was taken into PBS and centrifuged for 8 min at 350g. Red blood cell lysis was performed and three million cells were taken for antibody staining. Before staining, blocking (FACS buffer, 1%Rat Serum, 1%Fc Blocker) was done for 10 min at 4⁰c. Cells were incubated with primary antibodies (Pacific Blue-CD45 and APC-F4/80) for 20 minutes at 4⁰c. Cells were washed and filtered. Viable CD45+F4/80-  cells were sorted using a BD FACSAria™ III sorter. Cells were loaded onto a Chromium single-cell 3′v2 chip (10x Genomics) and libraries were prepared according to the manufacturer's protocol. Libraries were sequenced by Illumina HiSeq 2500 and the reads were mapped tomm10library (ensembl v98).