description: Protocols: GMSCs and PDLSCs were separated from the surface of the middle third of the root, and were digested with α-minimum essential medium (α-MEM, Gibco, Carlsbad, CA, USA) containing 3 mg/mL collagenase (type I) and 4 mg/mL dispase (both from Sigma-Aldrich, Billerica, MA, USA) for 15 min at 37 °C. Single-cell suspensions were obtained by passing the digested tissues through a 70-μm cell strainer (BD Bioscience, Franklin Lakes, NJ, USA). Then the cell suspensions were seeded into 10-cm culture dishes (Corning, USA) containing α-MEM supplemented with 15% fetal bovine serum (FBS, Hyclone, New York City, NY, USA), and 1 x GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2, and medium was changed twice a week. Cells were used at passages 3-5 for subsequent experiments. SP600125 (MedChemExpress LLC, Shanghai, China) were dissolved in dimethyl sulfoxide (DMSO). Unless otherwise indicated, cells were pre-incubated with serum-free medium containing same dose of DMSO (0.2%) or 10 μM SP600125 for 2 h. Here, we collected GMSCs and PDLSCs from systemically healthy patients due to the premolars and extracted for orthodontic reasons, and treated with tobacco smoke (G1, P1), E-cig smoke with original flavor (G2, P2), E-cig smoke with menthol flavor (G3, P3), E-cig liquid with original flavor (G4, P4), and E-cig liquid with menthol flavor (G5, P5) by the optimal conditions of LC50, and conducted RNA-seq to compare with untreated control (G0, P0). All followed iso smoking model. Total RNA of 5 x 107 GMSCs or PDLSCs were extracted using TRIZOL (Thermo Fisher Scientific). Nanodrop 2000 (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 (Agilent) were used to evaluate RNA concentration and quality. 5 μg of RNA from different experimental groups was prepared cDNA library using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following the manufacturer's instructions, and sequenced on an Illumina Hiseq 4000 platform.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-06-17
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-06-17
attributes.tag: External Id
attributes.value: SAMEA110006562
attributes.tag: INSDC center alias
attributes.value: Suzhou Ninth People's Hospital
attributes.tag: INSDC center name
attributes.value: Suzhou Ninth People's Hospital
attributes.tag: INSDC first public
attributes.value: 2022-06-17T00:17:35Z
attributes.tag: INSDC last update
attributes.value: 2022-06-17T00:17:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11797:G1_rep1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: dental papilla cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: premolar tooth
attributes.tag: sample name
attributes.value: E-MTAB-11797:G1_rep1