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Transcription from the locus of Nanog in mouse testes was further confirmed by the detection of testicular eGFP transcripts in transgenic Nanog eGFP-reporter mice (TNG) [3] (Fig. 1B).

Total RNA from mouse testes was isolated using TRIzol reagent (Invitrogen) and processed for Illumina gene expression analysis or QPCR.

Total RNA from mouse testes was first treated with calf intestinal phosphatase to dephosphorylate the 5'-ends of any truncated mRNA.

RNA isolated from highly purified samples of spermatocytes and spermatids from wild type and Hr6b knockout mouse testes was used for microarray analysis.

Similar analyses showed that a majority of DAZAP1 in adult mouse testes was also present in the cytoplasmic fraction (data not shown).

For testis piRNAs, 10 μg of total RNA from adult mouse testes was electrophoresed on an 18 % denaturing polyacrylamide gel with NEB miRNA markers, the gel stained with SYBR Gold, and the readily visible band migrating at 29 nt (range ~27-31 ~27-31cised.

For immunohistochemistry, mouse testes were fixed overnight in 4% paraformaldehyde (PFA)/PBS, dehydrated through graded ethanol, and processed for paraffin embedding.

Mouse testes were fixed overnight at 4°C within 4% paraformaldehyde in TBS, followed by subsequent incubation overnight at 4°C in 70% ethanol.

After institutional approval, mouse testes were obtained from fertile males that were housed at the Utrecht University Central Animal Facilities and the MRC Center Development in Stem Cell Biology, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh.

As a positive control, 5  μm thin sections of mouse testes were included.

Mouse testes were collected and digested by two-step enzymatic digestion procedure using collagenase and trypsin as described.