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Sample GSM2227770 Query DataSets for GSM2227770
Status Public on Dec 15, 2017
Title Primary human hepatocyte 5 days control replicate 2
Sample type RNA
Source name RNA from human primary hepatocytes
Organism Homo sapiens
Characteristics cell type: Primary human hepatocytes
treatment: exposed to 1% ethanol for 5 days daily
Treatment protocol PHH were exposed to 15 mM of VPA in a 24 hour repeat-dose testing regime. Medium was changed daily thereby providing a new dose of VPA to the PHH each day. A bi-phasic treatment regime was applied, combining a 5-day VPA exposure with a subsequent 3-days washout period during which the PHH were exposed to medium only. 1% Ethanol was used as solvent control.
Growth protocol Cryopreserved primary human hepatocytes (PHH) and culture media were purchased from Life Technologies (Bleiswijk, The Netherlands), unless otherwise stated.PHH were cultured in pre-coated multi-well plates in a 2-layer collagen sandwich.PHH were thawed for 1 min at 37°C. Three vials (one per donor) were poured in 50 ml Thawing Medium (CHRM CM7000) and centrifuged at 4°C for 10 min at 100g. The cell pellet was dissolved in plating medium (CM9000) at a concentration of 1*10E6 cells/ml. After seeding, PHH were incubated in the pre-coated multi-well plates for 4 hours at 37°C. Next, debris was removed by shaking and washing the cells twice with Williams’ Medium E medium. Subsequently, PHH were covered by a collagen layer (containing DMEM, NaOH and rat tail Collagen type 1 (BD biosciences, Breda, The Netherlands)), incubated for 30 min at 37°C, and covered with incubation medium (CM600, CM4000).
Extracted molecule total RNA
Extraction protocol At the end of treatment, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment.
Label Biotin
Label protocol cDNA was prepared using the Affymetrix One-Cycle cDNA synthesis kit (Affymetrix, Santa Clara). cDNA synthesis and labeling was performed according to the manufacturer’s rocedures. Subsequent labeling of the samples was conducted by synthesis of Biotin-labeled complementary RNA (cRNA) using the GeneChip IVT labeling kit (Affymetrix). Purified cRNA was quantified using a spectrophotometer, and unfragmented samples were checked on the Bioanalyzer. Subsequently, cRNA samples were fragmented for target preparation according to the Affymetrix manual and checked on the Bioanalyzer.Samples were stored at 20 C until ready to perform hybridization.
Hybridization protocol cRNA targets were hybridized on high-density oligonucleotide gene chips (Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays) according to the Affymetrix Eukaryotic Target hybridization manual. The gene chips were washed and stained using the Affymetrix Fluidics Station 450/250 and Genechip Operating Software
Scan protocol Chips were scanned by means of an Affymetrix GeneArray scanner
Data processing The Affymetrix CEL files were imported into R v2.15.0 [24] using the “affy” library within BioConductor (v2.9). Probesets were reannotated to EntrezGene genes using the BrainArray custom CDF v15.0 annotations. The quality of the arrays was assessed through box plots, fitPLM, NUSE, RLE, clustering/heat maps, PCA and correlation plots. No technically deviating arrays were detected. Probeset intensities were normalized using the RMA algorithm. For detecting differentially expressed genes, the “limma” library was used to construct a linear model containing coefficients for all factor combinations. The resulting p-values were FWER-corrected using the False Discovery Rate (FDR) approach.
Submission date Jul 07, 2016
Last update date Dec 15, 2017
Contact name Simone G van Breda
Phone 0031433882127
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 50
City Maastricht
State/province Limburg
ZIP/Postal code 6229 ER
Country Netherlands
Platform ID GPL16356
Series (2)
GSE84150 Induction of gene expression changes in primary human hepatocytes by valproic acid
GSE84250 Induction of changes in primary human hepatocytes by valproic acid

Data table header descriptions
VALUE RMA Log2 signal intensity

Data table
1_at 10.18646321
2_at 13.30461898
9_at 8.628769771
10_at 9.280622934
12_at 13.07498496
13_at 12.81689215
14_at 8.848237945
16_at 9.981259883
18_at 10.97639417
19_at 9.820606732
20_at 6.866752666
21_at 7.182126699
22_at 9.024523796
23_at 8.80211262
25_at 8.368386311
27_at 6.7345896
29_at 6.38474488
30_at 10.93168589
31_at 9.2552248
32_at 8.230101309

Total number of rows: 10752

Table truncated, full table size 217 Kbytes.

Supplementary file Size Download File type/resource
GSM2227770_C2.CEL.gz 4.5 Mb (ftp)(http) CEL
Processed data included within Sample table

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