Regulated Ire1-dependent decay of messenger RNAs in mammalian cells

Hollien, Julie; Lin, Jonathan H.; Li, Han; Stevens, Nicole; Walter, Peter; Weissman, Jonathan S.

We hybridized 5 µg each of a Cy5-labeled experimental sample and the Cy3-labeled reference pool to whole-genome mouse arrays at 65°C for 48 h. The arrays were produced in-house using the MEEBOChip platform. We extracted and processed image data using GenePix 6 (MDS Analytical Technologies), normalized each array to achieve a mean Cy5/ Cy3 ratio of 1.0, and removed spots containing low signal or poor signal uniformity. To simplify the analysis, we disregarded spots for which one or more samples did not pass these quality control measures and for which the SD of the three measurements was >30% of the mean. For the remaining spots (representing 25% of the total spots on the array), we calculated the log 2 ratio of DTT treated/untreated for each of the three replicates in the two cells lines. We then performed hierarchical clustering of the averaged data (Fig. S1 ).