GSM8322614: mandible,denervated,rep1,scRNA-seq; Mus musculus; RNA-Seq
Three Sham or Denervation 12-week-old male C57BL/6J wild-type mice were combined to extract single-cell suspensions.Three days after all 3 molars tooth extraction, mandibles were meticulously isolated under a stereomicroscope.Soft tissues and jaw bone around the incisor and behind condyle were cut off to obtain the region of tooth extraction socket tissues.These samples were rinsed with PBS and then finely minced (approximately 1mm3) on ice.The tissues were subjected to enzymatic digestion using a combination of 2mg/mL collagenase I (Biosharp) and 2mg/mL collagenase II (Biosharp) for 45 minutes at 37°C under gentle agitation.Post-digestion, the resultant mixture was filtered through a 70μm cell strainer, followed by centrifugation at 300g for 5 minutes.Discarding the supernatant, the cell pellet was resuspended in red blood cell lysis buffer (Miltenyi Biotec) to eliminate erythrocytes.Following a wash step in PBS supplemented with 0.04% BSA, the cell pellets were redissolved in the same buffer and subsequently sieved through a 40μm cell strainer.The dissociated single cells underwent viability staining with Calcein-AM and Draq7.This single-cell suspension was further refined using the MACS Dead Cell Removal Kit (Miltenyi Biotec). The BD Rhapsody system was employed for high-resolution single-cell transcriptomic profiling.A methodological strategy involving limited dilution was utilized to stochastically distribute the single-cell suspension across over 200,000 microwells.To ensure optimal interactions, oligonucleotide-barcode-carrying beads were introduced to saturation, facilitating a one-to-one association of each bead with a cell within a microwell.Within these microwells, cellular lysis was induced, allowing mRNA molecules to hybridize with the barcoded capture oligonucleotides present on the beads.Subsequently, these beads were consolidated into a single reaction vessel for reverse transcription and ExoI digestion processes.It is noteworthy that, during the cDNA synthesis phase, each cDNA fragment was uniquely labeled on its 5' end (corresponding to the 3' end of an mRNA transcript) with a unique molecular identifier (UMI) and a cell-specific barcode, denoting its cellular origin.For comprehensive transcriptomic analysis, the BD Rhapsody's single-cell whole-transcriptome amplification (WTA) protocol was adopted.This encompassed random priming and extension (RPE), RPE-targeted PCR amplification, and WTA index PCR.For accurate library quantification, tools such as the High Sensitivity DNA chip on the Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific) were utilized.The resulting libraries were subsequently sequenced using an Illumina sequencer, based in San Diego, CA, under a 150 bp paired-end run configuration.