Before seeding with LSK cells, UG26-1B6 stromal cells were plated into 0.1% gelatin-coated 12-well cell culture plates, (2x105 cells per well), grown to 100% confluence and irradiated at 30 Gy using a Mevatron KD2 (Siemens, Munich, Germany). Lin-Sca-1+c-Kit+ cells (LSKs) cells (>95% pure) cells were resuspended in stromal cell culture medium and seeded with stromal cells, ∼105 cells per well. Plates were incubated at 33◦C, 5% CO2 in air and 95% humidity for one to three days (Day1-Day3), respectively. In addition, in order to obtain Day 0 (uncultured; Day0; d0; 0h) cells, ∼2x105 UG26-1B6 stromal cells collected by trypsinization and washed. As an additional control, UG26-1B6 cells 24 h after changing the cell culture medium were also used (Day1 medium control; C).
Growth protocol
The midgestation-embryo-derived stromal clone UG26-1B6 (urogenital ridge-derived) cells were cultured on 0.1% gelatin-coated tissue culture plates in stromal cell medium (80% α-minimal essential medium (αMEM), supplemented with 15% fetal calf serum (FCS), 5% horse serum (HS), antibiotics penicillin and streptomycin (PenStrep; Gibco, Germany), and 10 μM β-mercaptoethanol (Gibco, Germany)) with 30% conditioned medium
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy Micro Kit (Quiagen Inc, Hilden, Germany), according to the manufacturer’s recommendation
Label
biotin
Label protocol
Biotin-labeled aRNA was obtained using MessageAmp aRNA Amplification Kit (Ambion, Austin, TX, US) and fragmented in RNA fragmentation reagent (Ambion, Austin, TX, US) by heating to 94◦C for 35 minutes.
Hybridization protocol
Biotinylated and fragmented aRNA was hybridized to the GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, US) using the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix, Santa Clara, US)
Scan protocol
standard Affymetrix protocol
Description
Gene expression data of UG26-1B6 stromal cells after three (d3; 72 h) day of culture co-culture with Lineage- Sca-1+ Kit+ (LSKs), biological rep1
Data processing
Microarray data analysis was performed using R /Bioconductor packages. Pre-processing of the microarray chips, including background correction, quantile normalization and summarization of the probe set values into expression measure was carried out using the GeneChip RMA (gcRMA) algorithm, as implemented into the gcrma package. The log2 scale data from gcRMA was used in statistical testing.
Submission date
May 13, 2015
Last update date
Sep 16, 2015
Contact name
Robert A.J. Oostendorp
Organization name
III. Medizinische Klinik und Poliklinik
Department
Klinikum Rechts der Isar Technische Universität München
