|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Jul 21, 2019 |
Title |
P 1 Healthy tissue stem cells- VitD |
Sample type |
SRA |
|
|
Source name |
stem cell enriched primary culture
|
Organism |
Homo sapiens |
Characteristics |
tissue: Healthy left colon gender: female age: 77 treatment: 1,25(OH)2D3 treatment: Vehicle
|
Treatment protocol |
Cells were treated with 1,25(OH)2D3 [100nM] or vehicle (EtOH) for 96 hours. The treatment was started 48 hours after passage and 1,25(OH)2D3 was renewed every 48 houres.
|
Growth protocol |
Normal and tumor culture medium was changed every other day and it was composed of 50% Advanced DMEM/F12, 50% Wnt3a-conditioned medium, 10 mM HEPES, 10 mM Glutamax, 10 mM nicotinamide (Sigma-Aldrich), 1x N2 (Thermo Fisher Scientific), 1x B27 (Thermo Fisher Scientific), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich), 1:100 Primocin, 0.1 μg/mL Noggin (Peprotech, NJ, USA), 1 μg/mL Gastrin (Tocris, Bristol, UK), 1 μg/mL RSPO1 (Sino Biological, Beijing, China), 50 ng/mL EGF (Peprotech), 0.02 μM PGE2 (Sigma-Aldrich), 1 μM LY-2157299 (Axon Medchem, VA, USA), 10 μM SB-202190 (Sigma-Aldrich) and 10 μM Y-27632 (Tocris). For passaging, we followed the protocol described by Jung et al. (2011) with modifications. Briefly, Matrigel-embedded organoids were collected using a scraper in a 50 mL conical tube and incubated with Cell Recovery Solution (Corning) for 30 min on ice and continuous shaking to remove Matrigel. After centrifugation at 1000 rpm 5 min at 4ºC, organoids were washed in washing buffer and centrifuged again. Next, organoids were incubated with disaggregation buffer (washing buffer solution containing 10 μM Y-27632 and 1 mg/mL Dispase (Thermo Fisher Scientific)) for 10 min at RT in orbital rotation. Right after 2 mM EDTA (Sigma-Aldrich) was added and the mixture was incubated for additional 5 min. Following this, the solution was homogenized by passing it through a 21G syringe 10 times, collected in a 15 mL conical tube, centrifuged at 1200 rpm for 5 min at 4ºC and washed twice in washing buffer in presence of 10 μM Y-27632. Pelleted cells were embedded in Matrigel and seeded on culture dishes.
|
Extracted molecule |
polyA RNA |
Extraction protocol |
Macherey-Nagel RNA extraction Kit was used in combination with TRIZOL. Both large and small RNA was extracted. RNA libraries were prepared for sequencing using standard Illumina protocols
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
1HD
|
Data processing |
Sequencing reads were aligned to the transcriptome with TopHat2. Novel transcript discovery was not attempted. TopHat was provided with known gene annotations and other transcript data obtained from Gencode (Version 26 - Ensembl 75) basic gene set for the GRCh37/hg19 human genome assembly. Gene expression level was calculated from TopHat alignments as the number of reads per gene computed using HTSeq using default settings and gene features as defined in the GRCh37.75 release of the human genome (gtf file). Differential gene expression analysis was performed with the Bioconductor DEseq2 package for the R statistical Software.
|
|
|
Submission date |
Jul 04, 2017 |
Last update date |
Feb 07, 2024 |
Contact name |
Alba Costales |
E-mail(s) |
albacostales@gmail.com
|
Phone |
0034647906865
|
Organization name |
IIB
|
Department |
cancer genetics
|
Lab |
231
|
Street address |
Arturo Duperier, 4
|
City |
madrid |
State/province |
madrid |
ZIP/Postal code |
28029 |
Country |
Spain |
|
|
Platform ID |
GPL11154 |
Series (1) |
GSE100785 |
RNA-Seq on the effect of VitaminD on colonic and tumor organoids |
|
Relations |
BioSample |
SAMN07313954 |
SRA |
SRX2982204 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|