title: GSM9168256: 10X scRNAseq 3M Spine WT; Mus musculus; RNA-Seq
library_name: GSM9168256
library_construction_protocol: Mice were euthanized, and the region of the vertebral column from vertebrae thoracic 11 (T11) to lumbar 2 (L2) were collected from Prss56-Nf1+/+ control and Prss56-Nf1fl/fl mutant mice at 3 months and 12-16 months of age. The vertebrae were cleaned from muscle, spinal cord, and dorsal root ganglia to avoid contamination from adjacent tissue. Before tissue processing, the collected vertebrae were observed under a fluorescent microscope to select the vertebrae with detectable tdTom+ signal. The tissues were minced using a scalpel and scissors and incubated in a digestion medium composed of 1mg/ml of Collagenase (C6885, Merck) and 2mg/ml of Dispase (D4693, Merck) in PBS for 20 min at 37°. Later 10ul/ml of 1% DNAse (WOLS02007, Serlabo, France) were added in the digestion medium and incubated for 10 min at 37°C. The supernatant was filtered and collected in sorting buffer composed of MEM-alpha and 10% FBS. The remaining tissue underwent a second step of digestion with 2ul/ml of 1% DNAse for 20 min at 37°C.  Collected cells were centrifuged at 40C for 10 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in sorting buffer and incubated for 30 min with antibodies for immune and endothelial cells CD11b-BV786 (BD Biosciences 740861), CD31- BV786 (BD Biosciences 740879), CD45-BV786 (BD Biosciences 564225) on ice and protected from light. After washing, cells were resuspended in sorting buffer, and Sytox Blue (S34857, Thermo Fisher Scientific) was added to stain dead cells. Using Influx Cell Sorter, the immune, endothelial, and dead cells were sorted and discarded, while Tom+ cells were collected. scRNA-seq libraries for the four datasets were prepared using the Chromium Single Cell Next GEM 3′ Library & Gel Bead Kit v3.1 (10x Genomics) according to the manufacturer's instructions.