Bioarray highyield rna transcript labeling kit t7

Double-stranded cDNA was synthesized from 5 lg total RNA using the Custom Superscript ds cDNA synthesis kit (Invitrogen, Carlsbad, CA) and used as a template for the preparation of biotin-labeled cRNA with use of the Bioarray HighYield RNA Transcript Labeling kit (T7) (Enzo Life Sciences, Inc, Farmingdale, NY). After fragmentation at 1 lg/ll according to the manufacturer's protocol, biotin-labeled cRNA (10 lg) was hybridized at 45 °C for 16-17 h to the RGU34A array (Affymetrix, Santa Clara, CA). Following hybridization, the arrays were washed, stained with phycoerythrin-streptavidin conjugate (Molecular Probes, Eugene, OR), and the signals were amplified by staining the array with biotin-labeled anti-streptavidin antibody (Vector Laboratories, Burlingame, CA) followed by phycoerythrin-streptavidin. The arrays were laser scanned with a GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions.

RNA was extracted from undifferentiated iPSCs; purified KP cells at days 3, 4, and 5, and CMs derived from KP cells at day 5 under serum condition (Figure S1B) using the RNeasy Mini Kit (Qiagen). Total RNA was reverse transcribed into cDNA via SuperScript II Reverse Transcriptase (Life Technologies). Samples were labeled with Cy3 using the BioArray HighYield RNA Transcript Labeling Kit (T7) (Enzo Life Sciences). Labeled cDNA was hybridized with Human Genome U133 Plus 2.0 Array by the GeneChip Hybridization Control Kit and the GeneChip Fluidics Station 450 (all Affymetrix) at 45 C for 16 hr. Arrays were scanned with the GeneChip Scanner 3000 7G according to the manufacturer's protocol and analyzed using GeneChip operating software (both Affymetrix). The raw data were normalized using the MAS5 algorithm (median score of the gene expressions: 500).