GSM5209371: PTH; Mus musculus; RNA-Seq
Cells from the cancellous bone region of the femur were isolated as follows.  Upon removal of the epiphysis, the metaphyseal bone region extending about 5 mm below the growth plate was dissected and crushed with mortar and pestle in FACS Buffer, followed by digestion with 0.25% collagenase type I (C0130, Sigma-Aldrich) in PBS (Gibco) for 20 min at 37 °C with gentle shaking. The cells were then centrifuged and re-suspended with FACS Buffer and filtered with a 70-μm cell strainer.  The tdTomato+CD31-CD45-Ter119- cells were sorted with Navios EX (Beckman Coulter).  Cell viability and number were determined with a cell counter (Biorad). 10x Genomics Chromium platform was used to capture and barcode cells to generate single-cell Gel Beads-in-Emulsion (GEMs) by following the manufacturer's protocol.  The oligonucleotides coating the gel beds enable mRNA capture by 30 bp oligo-dT and provide barcodes to index cells (16 bp) as well as transcripts (10 bp unique molecular identifier; UMI).  Following reverse transcription, cDNAs with both barcodes were amplified, and a library was constructed using the Single Cell 3' Reagent Kit (v2 chemistry) for each sample.  The resulting libraries were sequenced on an Illumina NovaSeq 6000 System.