GSM7719768: lateral meniscus, red zone; Sus scrofa; RNA-Seq
Tissues were rinsed in 5% Betadine in PBS solution followed by two rinsing in PBS, transported in MACS® Tissue Storage Solution on ice for up to 1 hour and processed immediately after arrival. The tissue was dissected away from the synovium, segmented into white and red zones, then cut into small pieces followed by enzymatic digestion, and disaggregation steps. In particular first incubation was carried out with 4mg/ml Pronase enzyme blend at 37oC for one hour followed by second in 3 mg/ml Collagenase P 37oC for another two hours. Mixture was filtered through 70 µm pluriStrainer® and centrifuged. Cell pellet was resuspended in 0,1% BSA in PBS and centrifuged for better removal of dead cells. Finally cells were filtered through 30 µm pluriStrainer® and resuspended in 1% BSA in PBS to prevent re-aggregation of cells. 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 libraries contain the P5 and P7 primers used in Illumina bridge amplification.