GSM8994829: 3w_3; Mus musculus; RNA-Seq
To isolate cells specifically from the metaphyseal (MP) region, the epiphysis was carefully removed at the midpoint of the growth plate using forceps. Residual chondrocytes from the growth plate were thoroughly removed under a stereomicroscope. The diaphysis was then trimmed approximately 2 mm (for 3- and 8-week-old Osx-CreERT2; mTmG mice) or 5 mm (for 5-week-old Osx-CreERT2; mTmG; LSL-tTA; Col1a1::TetO–c-Myc and Osx-CreERT2; mTmG; LSL-tTA; Col1a1::TetO–c-Myc; Trp53-LSL-R172H mice) distal to the growth plate. The harvested MP regions from distal femurs and proximal tibias were transferred to digestion buffer containing M199 with Hanks' salts (Gibco), 250 U/mL type I collagenase (Worthington), 250 U/mL type II collagenase (Worthington), 100 U/mL DNase I (1 mg/mL; Roche), 1 mg/mL poloxamer 188 (Sigma), 1 mg/mL BSA (Fujifilm), and 20 mM HEPES (Nacalai Tesque). The bones were placed in a mortar with 1 mL of digestion buffer and crushed using a pestle for at least 1 minute. The supernatant was collected into a 15 mL conical tube. This process was repeated until the bones appeared completely white (typically ≥3 times). The remaining bone fragments were pooled into the same tube and supplemented with digestion buffer to a final volume of 5 mL per sample. Samples were incubated at 37 °C for 30 minutes at 210 rpm. Following digestion, the supernatant was filtered through a 70 μm cell strainer into a 50 mL tube containing 5–10 mL of ice-cold cell suspension buffer [Ca²⁺/Mg²⁺-free PBS (Nacalai Tesque) with 2% FBS, 1 mg/mL poloxamer 188, and 100 U/mL penicillin/streptomycin (Gibco)]. Bone fragments were further digested with 3–5 mL of prewarmed digestion buffer, and the crushing–filtration process was repeated 4–5 times. Red blood cells were lysed using ACK lysis buffer (Gibco). Bone chips were removed, and single-cell suspensions were obtained by filtering through a 40 μm strainer for subsequent MACS preparation. Magnetic-activated cell sorting (MACS) was performed using LD columns (Miltenyi Biotec, Cat. No. 130-042-901) to deplete hematopoietic and erythroid cells with CD45 microbeads (Cat. No. 130-052-301) and TER119 microbeads (Cat. No. 130-049-901), following the manufacturer's instructions. Following MACS, single-cell suspensions were incubated on ice and in the dark for 30 minutes with the following fluorophore-conjugated antibodies (1:200 dilution; Invitrogen/eBioscience), prepared in cell suspension buffer: eFluor450-conjugated anti-CD31 (clone 390; Cat. No. 48-0311-82); eFluor450-conjugated anti-CD45 (clone 30F-11; Cat. No. 48-0451-82); eFluor450-conjugated anti-TER119 (clone TER119; Cat. No. 48-5921-82). Fluorescence-activated cell sorting (FACS) was performed using a FACS Melody system (BD Biosciences). mGFP-positive cells were isolated from the CD31⁻CD45⁻TER119⁻ fraction after dead cells were excluded using 7-AAD (1:200; BioLegend, Cat. No. 420403). Sorted cells were immediately collected into Buffer RLT Plus (Qiagen) and stored at −80 °C until RNA extraction. Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher), and RNA quantity was further confirmed using a Qubit fluorometer (Thermo Fisher). amples with an RNAintegrity number (RIN) >= 8, as determined by a Bioanalyzer (Agilent Technologies), were used for library preparation. RNA-seq libraries were generated from 200 ng of high-quality RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). The number of PCR amplification cycles was minimized to prevent amplification bias.