cell type: mESC, i-Mixl1 treatment: No Doxycycline time: day 4
Growth protocol
Differentiating i-Mixl1 ES cells cultured in the presence or absence of Doxycycline (DOX, 0.1 ug/ml, 3 replicates per treatment/time point).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from EBs harvested at day 2, 3 and 4 (DOX added 1 day after plating of ES cells) using RNeasy Mini columns (Qiagen). RNA (1 ug) was subjected to one round of linear amplification (RiboAmp System) to yield 10 ug of RNA
Label
Cy5
Label protocol
The RNA was indirectly labeled using amino allyl-dUTP('t Hoen, de Kort et al., 2003, Nucleic Acids Res. 31: e20), then conjugated with Cy3 or Cy5.
Hybridization protocol
The labeled RNAs were used to screen a 15K mouse developmental cDNA microarray(Tanaka, Jaradat et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97: 9127-9132).
Scan protocol
Hybridized microarrays were scanned using standard procedures (Tanaka, Jaradat et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97: 9127-9132).
Description
No Doxycycline
Data processing
Pairwise analysis of hybridization results for EBs cultured with or without DOX was performed for samples harvested on each day. Spotfire(R) software was used for data management and filtering.
