GSM7928287: Prx1 Cre; Ai9/+  mice; Mus musculus; RNA-Seq
Periosteal cells were extracted from femurs and tibias of Prx1 Cre; Ai9/+ and Prx1 Cre; Ai9/+; Piezo1 fl/fl mice at 8 week.  The femurs and tibias were capped by 5-10% Low Melting Point Agarose and then digested for 3 times (10 min, 45 min,45 min) with digestion buffer (1mg/ml collagenase, 2mg/ml dispase II and 1U/ml Dnase I in αMEM). The digested cells of the last two times were collected for sorting. The red blood cells were removed by RBC lysis buffer for 5 min (Beyotime, C3702). Then the Prx1 positive cells were sorted by BD FACS Aria Fusion. After thawing and resuscitation, the dissociated single cells were then stained with AO/PI for viability assessment using Countstar Fluorescence Cell Analyzer. The single-cell suspension was further captured using BD Rhapsody system. BD Rhapsody system was used to capture the transcriptomic information of the 2 samples-derived single cells. Single-cell capture was achieved by random distribution of a single-cell suspension across >200,000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation so that a bead was paired with a cell in a microwell. Cell-lysis buffer was added so that poly-adenylated RNA molecules hybridized to the beads. Beads were collected into a single tube for reverse transcription. Upon cDNA synthesis, each cDNA molecule was tagged on the 5′ end (that is, the 3′ end of a mRNA transcript) with a unique molecular identifier (UMI) and cell label indicating its cell of origin. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification workflow. In brief, second strand cDNA was synthesized, followed by ligation of the WTA adaptor for universal amplification. Eighteen cycles of PCR were used to amplify the adaptor-ligated cDNA products. Sequencing libraries were prepared using random priming PCR of the whole-transcriptome amplification products to enrich the 3′ end of the transcripts linked with the cell label and UMI. Sequencing libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). The library for each sample was sequenced by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.