GSM7849474: KO_CTR; Mus musculus; RNA-Seq
The aortas of each group of mice were pooled, cut into small pieces, and digested in 2.5 ml enzyme mix from Multi Tissue Dissociation Kit 2 (Miltenyi Biotec) prepared as described in the protocol provided by the vendor. The samples were placed at 37oC for 30 min with shaking (250 RPM). The suspension was gently pipetted every 15 min. Then the cells were filtered through a 70 μm strainer, centrifuged at 500 × g for 5 min, and resuspended in red blood cells lysis buffer (BioLegend). After 5 min, the cells were centrifuged at 500 × g for 5 min, washed with PBS, and resuspended in 300 µl of PBS containing 2% FBS and 200 ng/ml DAPI. To exclude dead cells and collect only the viable ones, the DAPI-negative cells were sorted with MoFlo XPD (Beckman Coulter) cell sorter. Cells isolated from meniscal zones were subjected to library construction with the use of Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics, #PN-1000269). Samples were processed according to the manufacturer's protocol. Firstly cell suspensions were divided into the single-cell format with a Chromium X Series controller (10x Genomics) that encapsulates single cells within nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common barcode. Incubation of the GEMs produced barcoded full-length cDNA from polyadenylated mRNA. After incubation, the GEMs were ruptured and the pooled fractions were recovered. Silane magnetic beads were used to purify the first-strand cDNA from the reaction mixture. Barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the size of the cDNA amplicons. Samples` indexes and TruSeq Read 2 were added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final librarieswere used in Illumina bridge amplification.