Total RNA was isolated from conditioned lin-/c-kit+ stem cells using the TRIzol reagent (Invitrogen, Germany) according to the manufacturer’s protocol. RNA was resuspended in DEPC–treated H2O (Sigma). RNA integrity and purity were electrophoretically verified by ethidium bromide staining and measurement of OD260/OD280 absorption ratios. Total RNA was purified from contaminating genomic DNA by a DNase I digestion (AMPD1, Sigma) using Qiagen RNeasy-Mini-Kit according to the manufacturer’s protocol. The quality of total RNA was checked by gel analysis using the total RNA Nano chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). Only samples with RNA index values greater than 8.5 were selected for expression profiling. RNA concentrations were determined using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Label
biotin
Label protocol
Biotin-labeled cRNA samples for hybridization on Illumina Mouse Sentrix-6 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine et al., 1992). In brief, 250 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the MessageAmp II aRNA Amplification kit (Ambion, Inc., Austin, TX). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water. cRNA Quality was checked using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).
Hybridization protocol
Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 50 ng cRNA/µl and unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes. After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals were developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays were dried and scanned.
Scan protocol
Microarray scanning was done using a Beadstation array scanner, settings were adjusted to a scaling factor of 1 and PMT settings at 430.
Description
Monoculture of mice lin-/c-kit+ bone marrow stem cells, Experiment 03
Data processing
Data extraction was done for all beads individually, and outliers were removed when MAD (median absolute deviation) was >2.5. All remaining data points were used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated. Normalisation with R/Bioconductor and beadarray package; Quantile normalisation and log2-transformation of raw data.
