GSM9251637: d7 callus from Sham; Mus musculus; RNA-Seq
Callus: Mice forelimbs were harvested 14 days after loading (n=3 for each group). Callus tissue was dissected and marrow cells were flushed out with PBS through the opening on both ends of callus. After cutting into small pieces, fracture tissues were digested with 1mg/ml Collagenase 1/Collagenase 2 (Sigma-Aldrich) and 2mg/ml dispase II (Roche). As soon as the cells were dissociated, cell suspension was centrifuged at 100g for 10 mins at 4°C. The cell pellet was resuspended and incubated in ACK lysing buffer (Quality biological, Cat #:118156101) for 10 minutes to remove red blood cells. Next, after centrifugation, cells were resuspended with PBS and filtered using 40μm cell strainer (Corning). In select experiments for CD45+ cell depletion, cells were processed using MojoSort™ Mouse CD45 Nanobeads (BioLegend) according to the manufacturer's protocol. Briefly, cells were resuspended in ice-cold MojoSort™ Buffer, filtered through a 70 μm cell strainer, and adjusted to 1 × 10⁸ cells/ml. Aliquots of 100 μl cell suspension (1 × 10⁷ cells) were incubated with 10 μl CD45 nanobeads on ice for 15 min. After washing with MojoSort™ Buffer, cells were subjected to two consecutive magnetic separations (5 min each) to ensure complete CD45+ cell depletion.  The dissociated cells were centrifuged again and the supernatant was removed until 100ul remained. for all samples, 10x Genomics Chromium standard protocol