title: RNA-seq of FACS isolated liver Kupffer cells one, three, six and twenty-four hours after systemically injecting mice with PBS or cKK LNPs carrying 0.3 mg / kg Cre mRNA
design: Cells were isolated 1, 3, 6 or 24 hours after injection with LNPs. Mice were perfused with 20 mL of 1X PBS through the right atrium. Liver tissues were finely minced, and then placed in a digestive enzyme solution with collagenase type I (Sigma Aldrich), collagenase XI (Sigma Aldrich) and hyaluronidase (Sigma Aldrich) at 37C at 550 rpm for 45 minutes. Cell suspension was filtered through 70 m mesh and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of , anti-CD45.2 (104, BioLegend), anti-CD68 (FA11, BioLegend). PBS-injected Ai14 mice were used to gate tdTomato populations for intravenous administration.The SMART-Seq v4 Ultra Low Input RNA method (Takara Bio, Japan) was employed for library construction, according to the manufacturers instructions. The amplified cDNA was purified by the Agencourt AMpure XP Beads (Beckman Coulter, USA) with 150 pg being processed further using Illuminas Nextera XT DNA library construction method according to the manufacturers instructions (Illumina, USA). The quantity and quality of the libraries were examined by Qubit DNA BR Assay Kit, Qubit 3.0 Fluorometer (Life Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies). Library sequencing was performed on an Illumina NextSeq platform to create paired-end reads with a length of 75 base pairs.The sequenced reads were trimmed and aligned to the mouse genome (mm10) using Isas analysis software 3.19.1.12, SAMtools 0.1.20, STAR aligner STAR_2.6.1a, Salmon quantification software 0.11.2, and Strelka Variant Calling software 2.9.9. Differential expression analysis was determined using DESeq2.