opossum #2 pachytene spermatocyte RNA-seq
Testes from Monodelphis domestica were obtained from animals culled from a colony maintained at the Texas Biomedical Research Institute. Populations of pachytene spermatocytes and round spermatids (purity 89-90%) were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C.  Unfixed aliquots of sorted cells were thawed on ice, and RNA isolation was performed using the RNEasy Mini kit according to the manufacturer’s instructions.  Genomic DNA was removed using gDNA eliminator columns supplied with the kit.  RNA-seq libraries were prepared using an Apollo 324 library prep instrument with supplied reagents (Integenx).