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Sample GSM2693220 Query DataSets for GSM2693220
Status Public on Jul 21, 2019
Title P 1 Tumor tissue stem cells-Control
Sample type SRA
 
Source name stem cell enriched primary culture
Organism Homo sapiens
Characteristics tissue: Tumor left colon
gender: female
age: 77
tnm: 300
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 1T
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 SAMN07313953
SRA SRX2982205

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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