Fig. 1. Transcriptome of Spga, Spcy, and Sptd. (A) Completeness of the germ-cell transcriptomes. The number of SAGE tags sequenced for each germ-cell library is plotted against the number of unique tags identified. The number of unique tags includes both singletons and gene tags. (B) Complexity of the germ-cell SAGE libraries. The number of each type of tags is indicated.

Fig. 2. Overlap of the transcriptome of type A spermatogonia, pachytene spermatocytes, and round spermatids. (A) The numbers in the Venn diagram represent the number of gene tags in the respective segments. The total number of unique gene tags in each library is indicated by the side of the name of the cell type. (B) Cell-specific transcripts are broken down into novel transcripts, uncharacterized cDNA, and known genes. The percentage of each category is indicated.

Fig. 3. Confirmation of splicing variation of Crem in germ cells. Orientation of the Crem transcript is indicated by 5′ and 3′. SAGE tags are shown as boxed nucleotide sequences. The putative polyadenylation signal associated with the SAGE tag is shown in red immediately 3′ of the SAGE tag. The poly(A) tail is indicated as (A)_n. AUUUA boxed with broken line represents the mRNA destabilizing sequence [26]. The number in front of the box indicates the number of copies of AUUUA in the sequenced franked by the putative polyadenylation signal immediately 5′ of the sequence and the 3′ SAGE tag. The QPCR primers used for generating the different amplicons are shown as arrows with the direction of amplification represented by the direction of the arrow. The names of the amplicons are shown underneath the line representing the DNA fragment amplified. The length of the lines is not proportional to the size of the actual size of the nucleotide fragment.

Statistics of the SAGE libraries

Type A spermatogonia were isolated from 6-day-old Balb/c mice whereas pachytene spermatocytes and round spermatids were isolated from 60-day-old mice using albumin gradient centrifugation and differential plating [42]. Total RNA was used for the construction of the SAGE libraries using the I-SAGE kit (Invitrogen Life Technologies, Carlsbad, CA). SAGE concatemers were sequenced using an AB PRISM 3100 Genetic Analyzer (PE Biosystems, Foster City, CA). SAGE2000 analysis software was used to extract SAGE tags from raw sequence data.

Comparison of two independent Spcy SAGE libraries

Total RNA from two independent preparations of Spcy containing comparable numbers of cells were used in the construction of SAGE libraries.

Tags encoding members of the Crem pathway identified in the germ cell SAGE libraries

Quantitative real-time PCR was performed to validate the differential expression of selected genes represented by the SAGE tag. Nucleotide sequence and copy number of the tags identified in each of the germ-cell SAGE libraries are shown in the SAGE column. The symbols of the amplicons produced using the primers described in Supplementary Information Table S11 and the means ± standard deviations of triplicate experiments are shown under the QPCR columns. All QPCR experiments were repeated using two different preparations of each type of cells. The QPCR results are normalized with the highest copy number of the SAGE tag.