title: GSM8618555: DEX 50; Mus musculus; RNA-Seq
library_construction_protocol: After four weeks mice were euthanized, and the hindlimbs were isolated and kept on medium at 4°C until further processing that day. The proximal left femur and the complete right femur were isolated, and muscle was removed. The bone tissue was cut in approximately 1 mm2 pieces, frozen viably and stored at -180°C for scRNA-seq. To obtain sufficient cell numbers for scRNA-seq, the femurs of the mice were pooled per treatment group. To increase the reliability of findings in the downstream analysis, such as differential gene expression and overrepresentation testing, vehicle animals of 2 studies with an identical study protocol were included, resulting in femurs of 20 mice in total pooled in the vehicle group. Next, to obtain a single-cell suspension, bone fragments were digested in two rounds of 30 minutes at 37°C, all centrifugation steps were done for 5 minutes at 300g. Digestion buffer consisted of Optimem (Gibco, cat no. 12634010), 100 U/mL DNase (Stemcell, 7469), 10 mM HEPES buffer (Gibco, 15630056), 1 mM CaCl2, 10 μM ROCK (Y-27632, Biogems, 1293823), and 0.1 mg/mL Liberase DL in the first digestion round (Sigma Aldrich, 5466202001) or 0.1 mg/mL Liberase TM in the second digestion round (Sigma Aldrich, 05401119001). After each digestion bone fragments were strained using a 70 μM cell strainer. Bone fragments were washed extensively at every step to collect cells. Collected cells were centrifuged and resuspended in 5 ml FACS buffer (magnesium-free PBS, 1% (vol/vol) BSA, 1 mM EDTA, 10 μM ROCK) and kept on ice. Red blood cell lysis buffer (Invitrogen) was added for 4 minutes at room temperature. Cells were washed, resuspended in 200 μl FACS buffer, and blocked for 15 minutes with 8 μg anti-CD16/CD32 antibody (Rat anti-mouse BD, 553079). Next, 4 μg CD45-FITC (Stemcell, 60161.2, Clone 2.4G2) incubated in the dark at 4°C for 30 minutes. Cells were washed twice with FACS buffer and resuspended in 1 mL FACS buffer. Sorting was performed with the Sony SH800 Cell Sorter, using a 100 μm nozzle chip. Droplet calibration was performed with Automatic Setup Beads (Sony, Cat. LE-B3001). Live and death cells were determined using 5 μM DRAQ5 (Biolegend) and 2.5 μM DAPI (Biolegend) staining. Gates were set using unstained samples as control, for DRAQ5 positive cells, DAPI negative cells, single cells, and CD45+ and CD45- cells. CD45+ and CD45- cells were sorted and subsequently combined in a 50/50 ratio of CD45+ and CD45- cells, and 45,000 cells per condition were subjected to droplet-based scRNA-seq using the 10x Genomics platform. The barcoding of individual transcripts from single cells, followed by cDNA amplification and library construction, were performed using 10X Genomics reagents and 10X Chromium Controller, according to the manufacturer's user guide nr. CG000315 (“Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index), RevC”).