Ribosome Sequencing of Testicular Spermatids in Mex3d Knockout and Wild-Type Mice
Ribosome Sequencing of Testicular Spermatids in Mex3d Knockout and Wild-Type Mice
Round and elongated spermatids were isolated and purified from adult mouse testes. Snap-frozen spermatids were cryoground into powder on dry ice, and then resuspended in ice-cold lysis buffer. After digest with RNase I, RNA was purified for ribosome-protected small RNA fragments via a 15% polyacrylamide TBE-urea gel. The gel debris was removed using a Spin-X column (Corning, 2628866), followed by ethanol precipitation. Purified RNA fragments (26-32 nucleotides) were resuspended in nuclease-free water and quantified using Qubit 2.0 Fluorometer (Invitrogen). A fraction of RNAs (10-200 ng) were used for cDNA library construction. After 3' polyadenylation, RNA was ligated to a linker at room temperature and purified using streptavidin magnetic beads. cDNA libraries were then generated through reverse transcription and PCR amplification, with no more than 14 cycles of PCR performed per group.