GSM8286084: SCC0104_pool2_KO_Multiseq; Mus musculus; OTHER
Mice (n=4 per genotype) were killed, perfused with cold PBS and whole aortas were dissected and harvested free of fat and connective tissue. Aortas were longitudinally dissected, and endothelial cells were removed manually using a cotton bud. Then, adventitial layers were manually removed after 10 min incubation in Collagenase Type IV (1mg/ml) and porcine pancreatic elastase (1 U/ml) in DMEM. Medial cell layers were then fully digested for 60 minutes at 37°C in Collagenase Type IV (2.5 mg/mL) collagenase IV and porcine pancreatic elastase (2.5 U/mL) in sterile DMEM. Aortic cell suspensions were labeled with CMO tags (10x Genomics) according to the manufacturer's instructions (Sm22Cre-Wnk1lox/lox aortas: CMO 4, 5, 6, 7; Sm22Cre+Wnk1lox/lox aortas: CMO 8, 9, 10, 11), washed and counted with viability assessed using Trypan blue (Sm22Cre-Wnk1lox/lox cells: 1000 cells/µl, 61% viability; Sm22Cre+Wnk1lox/lox cells: 837 cells/µl, 87% viability). Cells were loaded in the 10x Genomics Chromium with the aim to capture 10,000 cells per library using volumes according to 10x Genomics recommendations. Libraries were generated with the Chromium Single Cell 3' Reagents Kit v3 (10x Genomics) according to the manufacturer's instruction. Libraries were sequenced using Illumina NextSeq2000 with a P3 flow cell. Single cells were encapsulated into droplets with the Chromium™ Controller (10x Genomics). Transcripts captured in all cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 12 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer. MULTI-seq library: amplification was performed at 95°C for 5' followed by nine cycles at 98°C for 15''; 60°C for 30''; 72°C for 30'' and final elongation 72°C for 1' with 5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3' Small RNA RPI1 and RPI2 primer as i7 primer (Illumina). The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' SI-PCR primer as i5 primer (Illumina) was used for MULTI-seq library amplification. Following the final bead purification, both libraries were pooled as 90% mRNA library, 10%  MULTI-seq library before sequencing.