RNA-seq of Homo sapiens: periodontal ligament primary cell clone with fibroblastic phenotype
In this study, periodontal ligament (PDL) cells were collected from completely erupted third molars of healthy subjects (18-25 years old). Then, we purified PDL cell clones with potential to commitment with osteo/cementoblastic phenotype (C-O clones ) or clones committed to fibroblastic phenotype (C-F clones). RNA isolated from two C-O clones and two C-F clones cultivated in osteogenic induction medium, that was composed by Dulbecco’s modified Eagle medium high glucose (DMEM) supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/mL) (Gibco, USA), 50 mg/mL ascorbic acid (Sigma-Aldrich, China) 10 mM b-glycerophosphate (Sigma-Aldrich, USA), and 10-8 M dexamethasone (Sigma-Aldrich, USA), during 14 days were submitted to RNA-seq, and each clone was considered a biological replicate for C-O (G 13 and G48) and C-F group (G16 and G23). RNA-seq was performed using Illumina TruSeq RNA Sample Preparation kit v2 (Illumina, USA), according to the manufacturer’s instruction. For qRT-PCR, single-stranded complementary DNA (cDNA) was synthesized from 1 µg total DNA-free RNA using Transcriptor First Strand cDNA synthesis kit (Roche Applied Science, USA), following the manufacturer’s recommendations.