GSM5457082: Ldlr knockout aortic valve; Mus musculus; RNA-Seq
Mice were sacrificed in a CO2 chamber, and aortic valves were collected in Dulbecco's modified Eagle's medium (DMEM). Aortic valves were minced for a minute. Tissues were digested for 30 minutes with 1000 U/mL collagenase II and 90 U/mL DNase I in Ca2+/Mg2+ DPBS at 37 °C. The number of pooled mice in each experimental group was as follows: 30 mice for the C57BL/6J group (male, 12-week-old), 24 mice for the Ldlr-/- group, and 19 mice in the Apoe-/- group. Only live single-cells (PI negative single cells) were sorted using BD FACS Aria III. The single-cell suspensions were subjected to 3′ single-cell RNA sequencing aiming for target recovery of 5,000 cells (C57BL/6J; Ldlr-/-) and 3,000 cells (Apoe-/-) using Single Cell A Chip Kit, Single Cell 3' Library, and Gel Bead Kit V2, and i7 Multiplex Kit (10x Genomics). Solutions pertaining to 3′ chemistry and v2 of 10x Genomics were used to barcode individual cells. Libraries were sequenced on an Illumina HiSeq2500.