Comparison of small RNA profiling in sperm susceptible or not susceptible to epigenetic silencing of the LoxP site

During our attempt to engineer a conditional Kdm1a knockout using germline expressed Vasa-Cre transgene, we observed that the efficiency of LoxP recombination is dramatically reduced after the first recombination event occurs. This is due to a transvection event in which the recombination of one allele in one generation, blocks the recombination of the other allele in the following generation. Interestingly, this inhibition can be relieved by introducing an additional cross to a wildtype mouse between the first and the second Kdm1a recombination event. The recombination inhibition is therefore the result of epigenetic silencing that is erased once spermatogenesis is allowed to happen in the absence of Vasa-Cre. After excluding other possibilities, we considered the hyphotesis that a diffusible molecule targeting the LoxP site that could be deposited in sperm following the first Cre recombination event (original cross). This molecule should not be present anymore when the sperm is collected after the additional cross to wildtype (extra cross). To test whether this diffusible molecule could be a small RNA, we produced and compared small RNA libraries prepared from sperm derived from the original cross and from the extra cross. These samples are genetically identical but differ in their parental history as well as in the ability to recombine the second floxed Kdm1a allele in the following generation. The comparison aimed at finding potential small RNAs mapping to or close to the loxP site present only, or more abundantly, in the original cross. Unfortunately, we were not able to identify such small RNAs and we also could not find any consistent difference in small RNA profiles among the different samples. We therefore concluded that the transvection event is not connected to a diffusible small RNA accumulated in sperm. Sperm was isolated from the cauda epidydimis and washed with PBS. Trizol was used to extract RNA from the sperm samples with slight modificatiosn retain small RNAs. Sperm from each mouse was processed separately and treated as a replicate. Two biological replicates per condition were produced. For each sample, 250-1500ng of RNA were used for size-selection on a polyacrylamide gel. After elution from the gel we proceeded to library preparation.