GSM8228617: Library 2: Sm22Cre(+)Wnk1(lox/lox) aortas, GEX; Mus musculus; RNA-Seq
Mice under isoflurane anesthesia received an i.v. injection of 2µg anti-CD45.2-APC to label circulating leukocytes 5 minutes before sacrifice (cervical dislocation under isoflurane anesthesia). Mice were perfused with cold PBS and aortas were collected from Sm22Cre-Wnk1lox/lox (n=3) and Sm22Cre+Wnk1lox/lox (n=4) animals. Aortas were minced and digested for 1 hour at 37°C in RPMI containing 450U/ml collagenase I (Sigma-Aldrich C0130), 125U/ml collagenase XI (Sigma-Aldrich C7657), 60U/ml Hyaluronidase (Sigma-Aldrich H3506) for 1 hour at 37°C with agitation, washed in PBS/1% FCS and passed through a 70µm cell strainer to generate aortic cell suspensions. Cells were incubated for 20' at 37°C with 0.1µM Calcein-AM (BioLegend #425201) in PBS 1%FCS. Cells were subsequently incubated for 5 minutes with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101320, 10µg/ml), and labeled with 1:300 anti-Ter119 PE-Cy7 (BioLegend 116221) and 1:1000 Fixable Viability Dye eFluor™ 780 (ThermoFisher #65-0865-14, 1:1000). Cells from each aorta were washed and labelled with Multi-Seq oligos for sample multiplexing in PBS without FCS, washed, and cells from each genotype were pooled (Sm22Cre-Wnk1lox/lox pool of n=3 aortic cell suspensions; Sm22Cre+Wnk1lox/lox pool of n=4 aortic cell suspensions). After washing, viable Calcein-AM+Ter119-CD45.2-APC- cells were sorted using a BD FACS Aria III with a 100µm nozzle. Sorted cells were washed twice with PBS+0.04% ultrapure BSA, resuspended, counted with Trypan blue (final concentration 800 cells/µl, viability >90%) and 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 sequenced using Illumina NovaSeq6000 with a S1 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. OTHER: MULTI-seq (sample multiplexing for single-cell RNA sequencing using lipid-tagged indices)