GSM4677821: scRNAseq_WT; Mus musculus; RNA-Seq
4-week old mice were sacrificed and tibiae, femura, and calvariae were dissected. Soft tissue was removed through scraping, and the epiphysis was cut off. Bone marrow cells were flushed out with cold PBS using a syringe. Bones were cut into 1- to 2-mm lengths and subjected to 8 serial digestions of type I collagenase/EDTA. The final three collagenase fractions were collected by centrifuging the supernatant at 4ºC/300 rcf/8 min. Cell pellets were resuspended with 2% FACS buffer containing RNAse inhibitor and were filtered through a 100 µm cell strainer. tdTomato+/DAPI- cells were sorted (Sony SH800s Cell Sorter) into a new PCR tube containing 2% FACS buffer. Single cells were encapsulated into emulsion droplets using Chromium Controller (10x Genomics). scRNA-seq libraries were constructed using Chromium Single Cell 3' v3 Reagent Kit according to the manufacturer's protocol. Briefly, ~ 15,000 cells were loaded in each channel with a target output of 2,000 cells. Reverse transcription and library preparation were performed on C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad). Amplified cDNA and final libraries were evaluated on an Agilent BioAnalyzer using a High Sensitivity DNA Kit (Agilent Technologies). Libraries were sequenced with PE100 on the DNBSEQTM NGS technology platform (BGI, China) to reach ~ 450 million reads.