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Sample GSM1639697

Query DataSets for GSM1639697

Status

Public on Aug 08, 2016

Title

TP_037 Control DPSC Immortalized

Sample type

SRA

Source name

Human Dental Pulp Stem Cells

Organism

Homo sapiens

Characteristics

cell type: DPSC
genotype: Neurotypical Control
immortalized: Yes

Treatment protocol

20,000 cells/cm2 were seeded in poly-D-lysine coated 12 well plates in DMEM/F12 (1:1), 2.5% FCS, 100 U/ml penicillin and 100 μg streptomycin, and culture for 24 hours. Epigenetic reprogramming was performed by exposing the DPSCs to 10 μM 5-azacytidine in DMEM/F12 containing 2.5% FCS and 10 ng/ml bFGF for 48 hours. Neural differentiation was induced by exposing the cells to 250μM IBMX, 50μM forskolin, 200 nM TPA, 1mM dbcAMP, 10ng/ml bFGF, 10 ng/ml NGF, 30 ng/ml NT-3, and 1% insulin-transferrin-sodium selenite premix (ITS) in DMEM/F12 for 3 days. At the end of the neural induction treatment, the cells were washed with 1X PBS. Neuronal maturation was performed by maintaining the cells in Neurobasal A media supplemented with 1mM dbcAMP, 1% N2, 1% B27, and 30 ng/ml NT-3 and 1X Glutamax for 3 days.

Extracted molecule

total RNA

Extraction protocol

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent PGM

Data processing

These samples were run on Ion Torrent PGM.
These were aligned to the human genome (Enembl GRCh38.77) using TopHat version 2.0.10, Bowtie 2 version 2.1.0 and Samtools version 0.1.18
Reads were mapped to known genes using HTseq. Gene expression was analyzed using DESeq2 version 1.2.10
Genome_build: GRCh38.77
Supplementary_files_format_and_content: RPKM

Submission date

Mar 20, 2015

Last update date

May 15, 2019

Contact name

Quynh Tran

E-mail(s)

qtran@stjude.org

Organization name

St. Jude Children's Research Hospital

Department

Pathology