GSM1639693: TP_023 Control DPSC Immortalized; Homo sapiens; RNA-Seq
Control teeth were obtained through the Department of Pediatric Dentistry at the University of Tennessee Health Science Center (UTHSC). The UTHSC Institutional Review Board (IRB) approved this study and informed consent was obtained in accordance with IRB policy. Immediately following extraction, the teeth were placed in transportation media (DMEM/F12 with HEPES and 100 U/ml penicillin, 100μg/ml streptomycin). DPSC were isolated and culture as previously described in (Kiraly et al (2009) Neurochemistry International 55: 323-332) with slight modifications. Briefly, the pulp was minced and digested in a solution of 3 mg/ml Collagenase type I and 4 mg/ml Dispase II for 1 h at 37°C. Cells were seeded in poly-D-Lysine 12-well dishes and maintained under standard conditions (37°C, 5% CO2) in DMEM/F12 1:1, 10% fetal bovine serum (FBS), 10% newborn calf serum (NCS) and 100 U/ml penicillin, 100μg/ml streptomycin. Sub-confluent cultures were passaged regularly with 0.1μM HyQTase (HyClone). Total RNA was extracted from ~3 week cultured Neurons and DPSCs using the miRNeasy Mini kit from Qiagen. Total RNA was checked for quantity and quality on an Agilent Bioanalyzer 6000 pico chip and determined to have an RNA Integrity Number (RIN) of >8.0. NuGen amplified material was sheared on a Covaris S2 with a duty cycle of 10%, intensity of 5, 100 cycles/burst, and 6 X 60 sec cycles (total processing time 6 min).  500ng of this sheared dscDNA then used to prepare libraries for sequencing using the Ion plus Core Library Module for AB Library Builder System. Libraries were used from this point without amplification. Before sequencing, small aliquots of this material pooled and sequenced on an Ion Torrent PGM 314 chip. Barcode quantification data from the PGM were used to balance the barcodes for final pooling before sequencing. Following this final pooling the library pools were sized to a target size of 260bp on a Pippin Prep instrument. The sized libraries were examined on an Agilent High Sensitivity DNA chip, quantified using real-time PCR, and used to prepare spheres using a One-Touch 2 device. These spheres were then sequenced on an Ion Torrent Proton sequencer with a P1 chip.
GEO Accession
GSM1639693