ES cells were trypsinized (d0) and cultured in suspension to form embryoid bodies (EBs). Briefly, ES cells were transferred into Iscove's modified Dulbecco's Medium (IMDM) with 20% batch-tested FCS, nonessential amino acids (0.1 mM) and -mercaptoethanol (0.1 mM) (Invitrogen, Karlsruhe, Germany) in a 10 cm bacterial Petri dish and cultured on a shaker for 48 h. At d2, EBs were transferred into spinner flasks (Integra Cell Spin, IBS, Fernwald, Germany) and cultured for additional 7 days at 37°C, 5% CO2, and 95% humidity. Medium was exchanged at d5, d7, and d9; and at day 9 Puromyicn was added to select for cardiomyocytes. At d12, remaining cardiobodies were trypsinized to obtain a single cell suspension of cardiomyocytes (Cor.At cardiomyocytes). Cor.At cells were seeded onto a Fibronectin coated 10cm dish as described above, and cultured for 6h before extracting RNA.
Growth protocol
aPIG 44 cells were cultured on mouse embroynic fibroblasts in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with nonessential amino acids (0.1 mM), L-glutamine (2 mM), beta-mercaptoethanol (0.1 mM), LIF (ESGR) (500 U/ml), neomycin (6 µg/mL), and batch-tested fetal calf serum (FCS) (15% v/v) (all Invitrogen, Karlsruhe, Germany).
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted by using PeqGold RNApure (Peqlab Biotechnology, Erlangen, Germany) according to the manufacturer's instructions.
Label
biotin
Label protocol
Total RNA containing low molecular weight RNA was labelled using the flashtag RNA labeling kit (Genisphere, Hatfield, PA, USA) according to the manufacturer’s instructions.
Hybridization protocol
Sample was hybridized to a GeneChip® miRNA Array (Affymetrix, Santa Claraq, CA, USA) at 48°C and 60 rpm for 16 hours then washed and stained on Fluidics Station 450 (Fluidics script FS450_0003).
Scan protocol
Mircroarray was finally scanned on a GeneChip® Scanner 3000 7G (Affymetrix).
Description
mouse ES cells (D3, ATCC CRL 1934) transfected with the alpha-MHC-Pac-IRES-EGFP vector
Data processing
Data was normalized with RMA (Bioconductor, affy package). Normalization and probe set summarization was carried out on the mouse-specific probe sets only.
