cell line: H9 human embryonic stem cell line growth protocol: feeder layer time: 0 days
Treatment protocol
For differentiation, growth medium was replaced with RPMI with 0% serum, 100ng/mL activin A and 25 ng/mL Wnt3A for 1 day, followed by RPMI with 0.2% serum and 100ng/mL Activin A for 2 days, followed by RPMI with 2% serum and 100ng/mL Activin A for 1 days.
Growth protocol
H9 were plated on feeder layer of mouse embryonic fibroblasts (MEFs) and grown in DMEM/F12 supplemented with 20% knockout serum replacement, 4 ng/mL basic FGF, and 10ng/mL activin A
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions. The miRNA-enriched fraction of small RNAs was purified from total RNA by polyacrylamide gel electrophoresis using Ambion’s flashPAGE™ kits
Label
biotin
Label protocol
The 3' ends of the RNA molecules were tailed and biotin-labeled using the mirVana miRNA Labeling Kit (Ambion Inc., Austin, TX). The kit's dNTP mixture in the tailing reaction was replaced with a proprietary nucleotide mixture containing biotin-modified nucleotides (PerkinElmer, Waltham, MA).
Hybridization protocol
Hybridization, washing, staining, imaging and signal extraction were performed according to Affymetrix-recommended procedures, excet that the 20X GeneChip Eikaryotic Hybridization Control cocktail was omitted from the hybridization.
Scan protocol
Affymetrix GeneChip® 3000 7G scanner
Description
miRNA expression data from pluripotent hESCs
Data processing
The signal processing implemented for the Ambion miRCHIP is a multi-step process involving probe specific signal detection calls, background estimate and correction, constant variance stabilization and either array scaling or global normalization. For each probe, an estimated background value is subtracted that is derived from the median signal of a set of G-C matched anti-genomic controls. Arrays within a specific analysis experiment were normalized together according to the variance stabilization methods described by Huber et al. (Huber et al., 2002). Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-Test, with assumption of equal variance, was applied. One-way ANOVA was used for experimental designs with more than two experimental groupings or levels of the same factor. These tests define which probes are considered to be significantly differentially expressed, or significant, based on a default p-value of 0.001 and log2 difference > 1.
