GSM6652416: Skin organoid, Newborn, Day2, Control; Mus musculus; RNA-Seq
Cells were derived from the dorsal skin of neonatal mice within 24 hours of birth. The skin was floated in 0.25% trypsin solution at 4°C to separate into the dermis and epidermis. Epidermal cells were cut with scissors, pipetted, and filtered through a 70um cell strainer followed by centrifugation. The dermis was digested in 0.35% collagenase for 20 minutes, then filtered through a 70um cell strainer followed by centrifugation. The dissociated epidermal cells and dermal cells were mixed at a ratio of 1:9 and were dropped onto the upper chamber of a transwell culture insert, and the lower chamber was filled with 700ul DMEM/F12 (Corning) culture medium containing 10% FBS (Gibco). For adult cell culture, the growth factors were added from D0 to D4, followed by adding MMP14 recombinant protein from D4 to D7. PKC inhibitors were added throughout the cultivation period. The cells were cultured in a 5% CO2 at 37°C incubator, with the culture medium being changed every other day. Library was performed according to the manufacter's instructions (single cell 3' v3 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.