GSM7106552
Teeth from systemically healthy individuals (aged 21-27 years) with an indication for third molar extraction with intact periodontium, roots, and pulp, without periodontal inflammation, caries, and dental calculus, were obtained. Both the periodontal ligament (PDL) and dental pulp (DP) tissue samples were obtained using the protocol described in Iwata et al. (2010) and Yang et al. (2013). Briefly, teeth were washed with a solution containing 0.1% Amphotericin B 250 µg/mL + 1% PenStrep, (both from Gibco, Life Technologies, NY, United States) in α-Minimum Essential Medium (α-MEM, Life Technologies, NY, United States). PDL samples were obtained by scraping the middle third of the root. The DP tissue was collected after sectioning the tooth at the cementoenamel junction using a diamond bur (DandZ., Wiesbaden, Germany). Isolated pieces from PDL and DP were digested with 4 mg/mL dispase + 3 mg/mL collagenase Type I (Sigma-Aldrich, MO, United States) diluted in α-MEM for 1 hour (37 ˚C). Samples were periodically resuspended for 30 s at every 20 min interval. After digestion, cells were seeded in 6-well plates with specific culture media (α-MEM + 10% fetal bovine serum + 0.292 mg/mL glutamine + 1% PenStrep) and left in the incubator (37 ˚C with 5% CO2 and 95% air humidity) for 24 h. After this period, non-adherent cells and tissue fragments were washed out, and the medium was replaced every 48 h until the primary culture reached 75% confluency. Then, cells were split into 75 cm2 culture flasks until they reached passages 3 to 5. Illumina libraries were prepared following the manufacturer specifications of 10X GENOMICS Chromium System