Gli1-expressing stromal cells are highly reparative precursors of long-lived chondroprogenitors in the fetal murine limb

Single-nuclei RNA sequencing of cells dissociated from Hindlimb (femur and tibia, from RZ to PHT Region) from E16.5 embryos to identify how catch up grpwuth happens during injury and to to show transcriptional identities of contributors to chondrocytes in p21-overexpression induced injury in unilateral models. Pitx2-Cre/Cre; Col2a1-rtTA/+; Col2a1-eCFP/+ females were crossed with TigreDragon-p21/Dragon-p21 males and given Dox (1mg/ml in drinking water with 0.5% sucrose) at E12.5. The knee region (obtained via orthogonal cuts from the hypertrophic region of the distal femur to the hypertrophic region of the proximal tibia in skinned hindlimbs) of E16.5 Left and Right limbs from uCtl and Left-Cart-p21 samples (2 embryos of each genotype) were collected in DMEM with 10% DMSO and 5% calf serum, stored in a MrFrosty (ThermoFisher) at 4ºC for 10 min and then transferred to -80ºC overnight. Tissue lysis and nuclei isolation were done as described by L.G.M. in https://www.protocols.io/view/frankenstein-protocol-for-nuclei-isolation-from-f-5jyl8nx98l2w/v2, in two batches of four samples each (left and right knees from one control and one experimental embryo). Once ~5,000 nuclei per sample were collected, we proceeded with the 10x protocol described in https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/chromium-single-cell-3-reagent-kits-user-guide-v-3-1-chemistry, minimizing the time between nuclei preparation/sorting and chip loading. One chip per batch was used (4 channels in each case, to load left and right samples from one control and one experimental embryo).