Illumina NovaSeq 6000 paired end sequencing; Single-cell transcriptional profiling of primary human dental follicle and derived organoids
Single-cell transcriptional profiling of primary human dental follicle and derived organoids
Third molars were extracted at the 'Oral and Maxillo-Facial Surgery - Imaging & Pathology (OMFS-IMPATH)' unit of University Hospitals (UZ) Leuven after informed consent. The study was approved by the Ethics Committee Research UZ/KU Leuven (13/0104U). Dental follicle tissue was carefully isolated and collected in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 0.5% fungizone. Following short rinsing steps in 70% ethanol and phosphate-buffered saline (PBS; Gibco), tissue was minced into small (1mm2) fragments, and further dissociated using collagenase VI (3mg/ml) and dispase II (4mg/ml) for 2h at 37°C, while regularly pipetting up and down. Finally, single cells were passed through a 40µm cell strainer and collected in serum-free chemically defined culture medium (SFDM; DMEM/F12 1:1, 5 mg/l human transferrin, 5mg/l insulin Hybrimax from bovine pancreas, 35mg/L penicillin, 50mg/L streptomycin, 600µL/L ethanol, 50µl/L catalase, 1g/L NaHCO3, 0.5% BSA) and cells were counted using the Coulter Counter Z2 (Beckman Coulter). For organoids, Matrigel droplets were collected using ice-cold SFDM, and organoids were dissociated using TryplE (containing 1/1000 RI; Gibco) and mechanical trituration. Remaining organoid fragments were allowed to sediment and the supernatant containing single cells was centrifuged for 10 minutes at 190G. Supernatant was removed and the pellet containing single cells was collected in SFDM.  The collected dental follicle cell material was suspended in a mixture of SFDM and growth factor-reduced Matrigel in a 30:70 ratio, which was plated in 48-well plates at 20,000 cells per 20µL drop. After solidification, tooth organoid medium was supplemented. Rock inhibitor (RI; 10µM) was added the first day of seeding or passaging. Organoid cultures were kept at 37°C in a 1.9% CO2 incubator, and medium was refreshed every 2 to 3 days. Single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3' V2 Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 5,000 cells per library. Single cell libraries were prepared with the Chromium Single Cell 3' V2 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit from 10X Genomics.