title: GSM8472321: Fgfr3Ach KI mice; Mus; RNA-Seq
library_name: GSM8472321
library_construction_protocol: The mice were sacrificed, and tissues around the growth plate cartilage in the proximal tibia were dissected. Samples were cut into small pieces (1~2 mm) and incubated in digestion medium (preparation medium with 0.2 mg/ml LiberaseTM [Roche, Basel, Switzerland] and 2 kU/ml DNase Ⅰ [Merck, Rahway, NJ, USA]) for 4 h. The samples were then filtered using a 70 μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). For cell surface labeling, cell surface proteins were biotinylated as previously described36. Cells were resuspended in 1 ml of PBS supplemented with 1% fetal bovine serum (FBS) and 1 ng EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 4°C and washed. Next, the cells were stained with 0.6 μg/ml of TotalSeq (A0951-A0954 [BioLegend, San Diego, CA, USA]) for 20 min at 4°C, washed, and resuspended in PBS supplemented with 10% FBS and 1 μM Sytox Blue Dead Cell Stain (Invitrogen, Waltham, MA, USA) for 5 min at room temperature.  PE-positive and Sytox blue-negative cells were sorted using a FACS Aria II flow cytometer (BD Bioscience) with BD FACS Diva 9.0.1 (BD Biosciences) and suspended in PBS supplemented with 20% FBS. Library preparation and the first processing of paired-end fastq files were performed according to the workflow in the previously reported TAS-seq method (Shichino S, Ueha S, Hashimoto S, Ogawa T, Aoki H, Wu B, et al. TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq. Communications biology. 2022;5(1):602.). In brief, sorted cells were used to synthesize cDNA with BD Rhapsody Express Single-Cell Analysis System (BD Biosciences). cDNAs were amplified by the TAS-seq method at Immunogeneteqs Inc. (Noda city, Chiba, Japan) and sequenced with an Illumina Novaseq 6000 sequencer (Illumina, San Diego, CA, USA), using a Novaseq 6000 S4 reagent kit v1.0 or v1.5.
identifiers.namespace: GEO
identifiers.id: GSM8472321_r1