GSM3215434: Non-foam intimal macrophages rep 3 (Bulk RNA-seq); Mus musculus; RNA-Seq
The whole aorta (including the aortic sinus, arch, thoracic aorta and abdominal aorta) was carefully isolated, and the perivascular fat was removed. To remove the adventitia, the whole aorta was incubated with collagenase II (400U/ml)  with hyaluronidase (90 U/ml) at 37°C for 8 min, and then the adventitia was physically removed. The aortic tissue was cut into 2–5 mm pieces and incubated at 37°C for 70 min with gentle shaking in enzyme mixture containing DNase I (90 U/ml), collagenase I (675 U/ml), collagenase XI (187.5 U/ml), hyaluronidase (90 U/ml). Live foamy and non-foamy macrophages (PI-BODIPY493/503hiSSChiCD64+CD11b+, PI-BODIPY493/503loSSCloCD64+CD11b+) were sorted out using flow cytometry (BD FACSAria III). The sorted cells were quickly added to single cell lysis buffer. The cDNA libraries were prepared from cell lysates in 10X Single-Cell Lysis Buffer (Takara-Clontech) containing 5% RNase inhibitor. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per the manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator with the following parameters: duty cycle, 10; intensity, 5; cycles/burst, 200; and time, 180 seconds. The cDNAs were blunt ended, supplemented with an A base at the 3' end, and then ligated with Illumina sequencing adapters. The ligated fragments were amplified for 12 cycles using primers that incorporated unique index tags. The amplified fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases.
GEO Accession
GSM3215434