GSM6856041
We collected naturally exfoliated deciduous incisors from an 8-year-old child. The dental pulp tissue was gently isolated from the crown and minced into small pieces. Then the mixed solution, which was 3mg/ml collagenase type I and 4 mg/ml dispase in phosphate-buffered saline (PBS) buffer, was used to digest the pieces in 37°C for 1h. The single-cell solution of SHED was seeded into six-well plates and incubated in a 37°C 5%CO2 incubator. The culture medium was -MEM (Gibco) supplemented with 15% fetal bovine serum (Gibco), 1% glutamine (Gibco) and 1% penicillin/streptomycin (Gibco) and renewed every third day. The third-generation cells were digested with 0.25% trypsin and the single-cell suspensions obtained were applied for scRNA-seq. 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.