SAGE tag levels of photoreceptor-specific genes as measured by SAGE. Tag levels are normalized to 55,000.

Cumulative p-value distributions for each tag comparison made between SAGE libraries in the study. Monte Carlo analysis using binomial distribution was used to generate p-values for both observed and chance tag p-values. The software for carrying out this analysis is included in the SAGE 3.0.1 software package [18]. Differences are expressed as a cumulative fraction of the total tags for which comparisons could be made, which in this case is all tags present more than once in both libraries combined. Chance variation, the cumulative p-value difference expected for chance variation in sampling (values computed for comparison of SAGE libraries ONL1 vs ONL2 - the plot of chance cumulative p-value differences is essentially the same for each library comparison considered); sampling, ONL1 vs ONL2 (same library, different tags); ditag, P2.5A vs P2.5B (same ditag ligation, different libraries); mRNA, crx^+/+A vs crx^+/+B (same starting mRNA, different libraries); 44-year-old vs 41-year-old, libraries from peripheral retina from different similarly aged males; 88-year-old vs 44-year-old, libraries from peripheral retina from individuals who differ in age and sex; littermates, P6.5A vs P6.5B (libraries made from retinas of three P6.5 C57B/6 mice vs libraries made from retinas of three littermates); environment, hypothalamus A vs hypothalamus B (libraries made from hypothalami of 3 8-week-old male C57B/6 mice housed in Boston, MA vs libraries made from hypothalami of 20 8-week-old male C57B/6 mice housed in Melbourne, Australia); retina vs hypothalamus, libraries from adult male C57B/6 mouse retina vs adult male C57B/6 mouse hypothalamus; retina vs 3T3 cells, libraries from adult male C57B/6 mouse retina vs mouse 3T3 fibroblast cell line.

SAGE tag abundance levels for tags in the 41-year-old peripheral retina SAGE library (retina kept at room temperature 5 h post-enucleation) and the 44-year-old peripheral retina library (retina kept at room temperature 45 min post-enucleation) for tags that differed at p ≤ 0.005; 139 such tags are shown.

Virtual Rot analyses. (a) Virtual Rot analysis of hypothalamus library A. This plots the cumulative fraction of tags in the library against the total tags present for each tag-abundance level. The boundary between moderate- and low-abundance transcripts is set at the fraction of tags present only once in each library, as tag number (approximately 55,000) is not sufficient to comprehensively cover every cellular mRNA (around 300,000 molecules of mRNA/cell). (b) Virtual Rot analysis of adult retina library, carried out by the same method as (a). (c) mRNA abundance classes derived from the virtual Rot analysis in (a) and (b), compared to an actual Rot analysis of mouse whole-brain polysomal mRNA obtained from [11]. ND, not determined.

SAGE tag levels of cell-cycle regulatory genes as measured by SAGE. Tag levels are normalized to 55,000.

Examination of tag levels and fold-change in each of the SAGE library comparisons considered in Figure 4. The data is plotted in a log scale in rotated coordinates, where log₂(xy) serves to indicate tag levels in the two libraries (where x = tag levels in SAGE library A, and y = tag levels in SAGE library B) and log2(y/x) serves to indicate fold changes in tag levels. Tags with a count of zero in either library were omitted from the analysis. The dark lines indicate the two standard deviation confidence limit calculated for the distribution of tags in Figure 5c (that is, same starting mRNA, different SAGE libraries) using a locally weighted smoothing technique based on the standard deviations in the neighborhood of each point. This is done to allow ready comparison of the observed tag differences between SAGE libraries to the baseline level of variability expected from SAGE library construction. (a) Sampling, ONL1 vs ONL2 (same library, different tags); (b) ditag, P2.5A vs P2.5B (same ditag ligation, different libraries); (c) mRNA, crx^+/+A vs crx^+/+B (same starting mRNA, different libraries). (d) 44-year-old vs 41-year-old, libraries from peripheral retina from different similar-aged males; (e) 88-year-old vs 44-year-old, libraries from peripheral retina from individuals that differ in age and sex; (f) littermates, P6.5A vs P6.5B (libraries made from retinas of three P6.5 C57B/6 mice vs libraries made from retinas of three littermates); (g) environment, hypothalamus A vs hypothalamus B (libraries made from hypothalami of 3 8-week-old male C57B/6 mice housed in Boston, MA vs libraries made from hypothalami of 20 8-week-old male C57B/6 mice housed in Melbourne, Australia); (h) retina vs hypothalamus, libraries from adult male C57B/6 mouse retina vs adult male C57B/6 mouse hypothalamus; (i) retina vs 3T3 cells, libraries from adult male C57B/6 mouse retina vs mouse 3T3 fibroblast cell line. See Table 1 and Materials and methods for more information about these SAGE libraries.

SAGE tag abundance levels for tags in the P6.5A SAGE library (generated from tissue stored for 1 week at -80°C) and the 44-year-old peripheral retina library (generated from tissue stored for 6 months at -80°C) for tags that differed at p ≤ 0.005; 135 such tags are considered.