GSM5293432: WT3; Mus musculus; RNA-Seq
Preparation of a single cell suspension of aortic cells was performed using a previously described enzymatic digestion protocol9. Briefly, the isolated whole aorta was finely cut and incubated in 1X Aortic Dissociation Enzyme Solution (125 U/mL collagenase type XI, 60 U/mL hyaluronidase type 1-s, 60 U/mL DNase I, and 450 U/mL collagenase type I) for 1 hour at 37°C. The cell suspension was strained through a 30 μm filter, treated with ACK lysis buffer for 5 minutes at room temperature, and washed twice with PBS. The cells were resuspended in 0.4% BSA-PBS at a final concentration of 8×105 cells/mL. Single cells were processed through the GemCode Single Cell Platform using GemCode Gel Bead, Chip and Library Kits (10X Genomics) as per the manufacturer's protocol. In brief, single cells were sorted into 0.4% BSA–PBS solution. 9,000 cells were added to each channel. The cells were then partitioned into Gel Beads in emulsion in the GemCode instrument, where cell lysis and barcoded reverse transcription of RNA occurred, followed by amplification, shearing and 5′ adaptor and sample index attachment. Libraries were sequenced on an Illumina Hiseq 2500. 10x genomics single cell v2