title: GSM8548318: dmm surgery, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM8548318
library_construction_protocol: For scRNA-seq, intact long bones were isolated, and attached soft tissues were removed using forceps, and dissociated periosteum cells and epiphysis articular cartilage cells with enzymatic digestion solution: 3 mg/ml type I collagenase (Worthington, LS004197), 4 mg/ml Dispase II (Sigma, 04942078001) and 1 U/ml DNase I (Sigma, D4527) in HBSS with Ca2+ and Mg2+. After four rounds of enzymic digestion in 37°C water bath (20 min per round), cells were resuspended in staining buffer (Ca2+ and Mg2+ free HBSS with 2% FBS) with 2 mM EDTA to stop the digestion for flow cytometry. Dissociated cells were stained by standard protocols with the following antibodies (eBioscience, Shanghai, China). allophycocyanin (APC)-conjugated CD31 (390), CD45 (30F-11), Ter119 (TER-119). DAPI and CalceinAM were used for viable cell gating. For RNA-seq, Intact long bones were isolated, and attached soft tissues were removed using forceps, and dissociated epiphysis articular cartilage cells with enzymatic digestion solution: 3 mg/ml type I collagenase (Worthington, LS004197), 4 mg/ml Dispase II (Sigma, 04942078001) and 1 U/ml DNase I (Sigma, D4527) in HBSS with Ca2+ and Mg2+. After four rounds of enzymic digestion in 37°C water bath (20 min per round), cells were resuspended in staining buffer (Ca2+ and Mg2+ free HBSS with 2% FBS) with 2 mM EDTA to stop the digestion for flow cytometry. Dissociated cells were stained by standard protocols with the following antibodies (eBioscience, Shanghai, China). allophycocyanin (APC)-conjugated CD31 (390), CD45 (30F-11), Ter119 (TER-119), Procr (eBio1560), PE/Cy5-CD105 (MJ7/18). DAPI and CalceinAM were used for viable cell gating. For scRNA-seq library construction, harvested cells were resuspended at 1 × 103 cells/mL and loaded on Chromium Controller to obtain single cells (10× Genomics) according to the manufacturer's protocol of Beijing SeekGene BioSciences Co., Ltd (Beijing, China). Chromium Single cell 3' Library and Gel Bead Kit V2 (10× Genomics, PN120237) was used to generate single cell gel beads in emulsion (GEM). The captured cells were lysed, and the released RNA was reverse-transcribed with primers containing poly-(d)T, barcode, Unique Molecular Identifiers (UMIs) and read 1 primer sequence in GEMs. Barcoded cDNA was purified and amplified by PCR. The adapter ligation reaction was performed to add sample index and read 2 primer sequence. After quality control, the libraries were sequenced on Illumina Novaseq 6000 platform in 150 bp pair-ended manner.