GSM8774093: PDL; Mus musculus; RNA-Seq
All ｍaxillary molars were extracted using hooked-end forceps under triple anesthesia (medetomidine hydrochloride 0.75 mg/kg [Domitol; Nippon Zenyaku Kogyo Co., Ltd], midazolam 4.0 mg/kg [Dormicum; Sandoz K. K.], and butorphanol 5.0 mg/kg [Vetorphale, Meiji Seika Pharma Co., Ltd.]). After surgery, euthanasia was performed by cervical spine fracture-dislocation under anesthesia. The extracted 6 molars were incubated with 2 mL of collagenase/protease/DNase mixed buffer (2 Wunsch units of Liberase™ [Roche], 0.5 mg/mL Pronase [Roche], 1/2000 benzonase [Sigma-Aldrich], and 5 mM MgCl2 [Sigma-Aldrich]) modified from a previous report 49) at 37 °C, 2000 rpm for 1 h in a shaking incubator (ThermoMixer® C, Eppendorf). The incubation buffer was collected into a tube with a 35-μm filter cap (Falcon, Corning Inc.) containing 1 ml bone buffer (2% fetal bovine serum, 0.5 mM ethylenediaminetetraacetic acid [EDTA, Thermo Fisher Scientific], 25 μg/ml DNase [Worthington Biochemical Corporation], and 5 mM MgCl2 in phosphate-buffered saline), and centrifuged at 4 °C,  280 g for 10 min. The cell pellet was treated with a hemolysis buffer (ACK Lysing Buffer, Thermo Fisher Scientific) containing 1/2000 benzonase (Sigma-Aldrich) and 5 mM MgCl2 for 3 min on ice. The cells were incubated with APC-conjugated rat anti-CD45 antibody (clone: 30-F11, Thermo Fisher Scientific) (1:100) and APC-conjugated rat anti-Ter119 antibody (clone: TER-119, Thermo Fisher Scientific) (1:100) for 30 min on ice. Cell sorting experiments were performed using a FACSMelody (BD Biosciences) equipped with FACSChorus software (BD Biosciences). Dead cells and debris were excluded using FSC, SSC, and SYTOX Red Dead Cell Stain (Thermo Fisher Scientific) staining profiles. CD45, Ter119-negative living stromal cells were collected and used for scRNA-seq analysis. Because our pre-experiment suggested that the frequency of Lepr-cre-Tom+ cells in the sorted stromal population were too low to perform their gene profiling analysis, we added additional CD45, Ter119-negative living Lepr-cre-Tom+ cells to the sorted CD45, Ter119-negative living stromal sample. Single cell suspension was prepared according to the manufacture's instruction of 10x Genomics and filtered through a 40-μm cell strainer. Isolated cells were prepared for single cell suspension using a 10x Chromium Controller (10x Genomics). Single Cell 3' Library kit v3.1 were used for cDNA library preparation and the protocol was followed by 10x Genomics. The quality of the libraries was checked using a BioAnalyzer 2100 (Agilent) and LabChip （PerkinElmer）. Sequencing was performed using NovaSeq 6000 (Illumina) with read１:28 bp、index：8 bp、read2：91 bp.