GSM8229614: C7-T1 DRGs d56 1st library; Mus musculus; RNA-Seq
DRG: Retrogradely labeled whole DRGs at C7-T1 from injected side were harvested from 4 time points (uninjured, 1-, 14- and 56-days post-fracture). The dissection and dissociation were conducted using previously published protocol [9], with minor modifications. Briefly, 18 DRGs from 6 mice were collected into ice-cold PBS. The DRGs then were digested with 1.8ml digestion solution with Papain (25U/ml, Worthington, Cat #:LS003126), Collagenase A (20mg/ml, Sigma-Aldrich, Cat #:10103586001), Dispase II (20mg/ml, Roche, Cat #:61926600) and BSA (0.5%, Sigma-Aldrich, Cat #:A7979) at 37 °C. 30ul of Collagenase A/Dispase II (20mg/ml) was added depending on the dissociation progress. Once DRGs were dissociated, the cell suspension was filtered using a 40μm cell strainer (Corning), diluted with 3ml aCSF (Tocris Bioscience) and centrifuged at 100g for 10 mins at 4°C. After removing the supernatant, the pellet was resuspended in 0.5ml aCSF and 0.5 ml complete Neurobasal medium. The cell suspension was then carefully layered on top of an Optiprep gradient (90ul Optiprep density solution (Sigma-Aldrich) + 455ul aCSF + 455ul complete Neurobasal). The gradient was centrifuged at 100g at 4°C for 20 mins. After centrifuging, the supernatant was removed until 100ul remained. 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). The dissociated cells were centrifuged again and the supernatant was removed until 100ul remained. for all samples, 10x Genomics Chromium standard protocol