Isolation and Analysis of Hepatic CD11c+ Cells

Livers were collected at the indicated times after transcardial perfusion to clear organs of red blood cells. Liver samples were digested in collagenase IV (MP Biomedicals, LLC) at 37 °C for one hour and then processed on a 70 µm nylon cell strainer (Falcon) into a single cell suspension. Liver samples were resuspended in 30% Percoll solution and centrifuged at 750g for 20 min without brake. Supernatants were discarded and pellets were resuspended in RBC Lysis Buffer (BioLegend) for 5 min. Cells were then washed and ready for magnetic separation. The CD11c MicroBead kit UltraPure (Cat#130-125-835, Miltenyi Biotec) was then used according to the manufacturer’s conditions in order to isolate CD11c⁺ cells. Total RNA was isolated using MicroRNAeasy (Qiagen, Valencia, California) from freshly isolated hepatic CD11c⁺ cells. 10 ng of input RNA was used to produce cDNA for downstream library preparation. Samples were sequenced on a NextSeq 500 (Illumina) system as previously described^{63 (link)–65} . Raw reads were aligned, normalized, and further analyzed using Partek Genomics Suite software, version 7.0; Partek Inc., St. Louis, MO, USA. Pathway analysis was performed using the Qiagen IPA software.