Illumina HiSeq 4000 sequencing; Single cell RNA sequencing of myeloid cells from the aortas of ApoE-/- mice fed a high fat diet for 12-16 weeks
Single cell RNA sequencing of myeloid cells from the aortas of ApoE-/- mice fed a high fat diet for 12-16 weeks
Single cells from aortas were digested in 5% FBS/RPMI containing 450 U/ml collagenase I, 125 U/ml collagenase XI 60 U/ml hyaluronidase and 60 U/ml DNase I for 50 minutes at 37°C. Cells were retrieved by passing tissue pieces through a 70 um cell strainer and were pooled from 9 mice in each experiment. Unspecific binding was blocked by incubating cells with murine anti-CD16/32 antibody. Cells were stained with antibodies against CD45, CD11b and CD64 and LIVE/DEAD viability dye. Live CD45+ CD11b+ (two experiments) or live CD45+ CD11b+ CD64low-high (one experiment) cells were sorted for sequencing.  Library generation for 10x Genomics v2 chemistry was performed following the Chromium Single Cell 3ʹ Reagents Kits (User Guide: CG00052).  Quantification of cDNA and library construction was performed using a Qubit dsDNA HS Assay Kit (ThermoFisher Scientific) and high-sensitivity DNA tapestation (Agilent).