GSM7664987: bone,1797 02; Sus scrofa; RNA-Seq
For tissue digestion and single cell isolation, we used 3mg/ml typeI collagenase diluted with serum-free DMEM. Briefly, we isolated intact regenerated bone tissues and then kept them within pre-cold PBS. And then tissues were chopped into 3-5um sized pieces and then subjected to digestion under 37℃ for 1h. After digestion, all suspension were collected and erythrolysised for following experiments. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.