GSM8970269: Cranial bone deffect, Gel100, 7 days, biol rep 5; Mus musculus; RNA-Seq
Upon harvest at week 1, cells from the five mice were pooled together for each material composition. Scaffolds from different groups were washed with DPBS and digested in an enzyme mixture containing 0.1 mg/mL Liberase TM (Sigma-Aldrich), 0.2 U/mL Chondroitinase (Sigma-Aldrich ABC C2905), and 100 U/mL Hyaluronidase (Sigma-Aldrich H3506) for 30 minutes at 37°C on a shaker. The digested scaffolds were then passed through a 70 μm cell strainer, with any remaining tissue in the strainer being minced using a syringe plunger and washed with DPBS containing 2% FBS. Red blood cells were removed with red blood cell lysis buffer (BioLegend). Cell viability was assessed via flow cytometry, and the viable cells were sorted for library preparation Approximately 10,000 viable cells were loaded into a 10x Chromium Controller (10x Genomics) to generate single-cell gel beads-in-emulsion (GEMs). The GEMs were processed to reverse transcribe the cells' RNA, producing cDNA, which was then amplified. This amplified cDNA was used to construct the single-cell library using the Chromium Single Cell 3' Reagent Kits v3 (10x Genomics) according to the manufacturer's protocol. The libraries were quantified with a Qubit fluorometer, and their quality was assessed using an Agilent Bioanalyzer. The resulting libraries were sequenced on an Illumina NovaSeq 6000 platform to achieve the desired sequencing depth.