GSM1639689: TP-078 Deletion DPSC; Homo sapiens; RNA-Seq
Control teeth were obtained through the Department of Pediatric Dentistry at the University of Tennessee Health Science Center (UTHSC). Teeth from AS deletion and idic15 subjects were shipped from various locations by the parents of children with these conditions. 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 subjected to analysis on a Agilent Bioanalyzer 6000 pico chip and determined to have a RIN of >8.0. 5ng of total RNA then used to prepare dscDNA using the NuGEN Ovation V2 kit. 500ng of this dscDNA then used to prepare libraries for sequencing using the AB Library Builder™ Fragment library Kit for 5500 Genetic Analysis Systems on a Library Builder system. Libraries were amplified 7 cycles before 5500 Wildfire primers added by 5 cycles of fusion primer amplification as described in the 5500 Wildfire manual (Applied Biosystems). Before sequencing, small aliquots of this material pooled and sequenced on an Ion Torrent PGM 314 chip after additional amplification with PGM fusion primers. Barcode quantification data from the PGM was used to pool the samples with Wildfire primers equally. Following this final pooling the library pools were examined and quantified on an Agilent high sensitivity DNA chip, immobilized on flow cells for the 5500 Wildfire instrument, and sequenced (50bp reads) on this instrument.
GEO Accession
GSM1639689