strain: 129-Sv cell line: J1 cell type: embryonic stem time: 4 days differentiation: differentiated Flk1+ WT ES cells
Biomaterial provider
We thank Nancy Speck (Harvard Stem Cell Institute, Cambridge, USA) for giving the Runx1-/- ES cell line to us, as well as Stuart H. Orkin (Abramson Family Cancer Research Institute, Philadelphia, USA) for giving the Tal-1-/- ES cell line to us.
Growth protocol
Undifferentiated ESCs were plated at on pre-seeded confluent, non-irradiated OP9 stromal cells (day 0) and cultured in αMEM supplemented with 20% FCS (Gibco-Invitrogen). On day 4, 5, and 6 of differentiation “in vitro”, cells were harvested by trypsinization and FACS purified for Flk-1+ cells (98% purity).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digest. The amount, purity, and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).
Label
Biotin
Label protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG430Av2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Hybridization protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG430Av2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Scan protocol
Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software from Affymetrix.
Description
Gene-expression profiling of three biological replicates was performed at day 4 during the differentiation process of WT J1 ESCs (3 samples). Samples were analyzed using mouse Genome 430-2 arrays (Affymetrix).
Data processing
Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 chips of one group were compared to each 3 chips of any other group, and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 9 SLR values of Experiment group vs. Baseline group and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).
