Project description:Pachytene spermatocytes were isolated from adult wild-type and Hsf5 knockout mouse testes using STA-PUT velocity sedimentation. Single-cell suspensions were prepared by enzymatic digestion of seminiferous tubules with collagenase and trypsin. Cells were loaded onto a 4%–2%–0.5% BSA gradient and allowed to sediment for 1 hour and 45 minutes at 4°C. Fractions were collected, and pachytene spermatocytes were identified by SYCP3, γH2AX, and DAPI staining under microscopy. Enriched pachytene cells were pooled and processed for RNA-Seq.
Project description:piRNA-deficient Maelstrom (Mael) null mice are characterized by a strong upregulation of LINE-1 (L1) retrotransposon in meiotic spermatocytes. This defect turns out in the accumulation of L1 RNA and ORF1p in their cytoplasm and the formation of prominent ribonucleoprotein aggregates. We used 3-months-old Mael-/- male mice to characterize the RNA present in those ORF1p aggregates. To favor the isolation of complexed versus free ORF1p protein, we first fractionated Mael-/- testis extracts (that we refer to as TOTAL) by sucrose gradient ultracentrifugation, in the presence of EDTA. We then pooled the sucrose fractions where ORF1p macromolecular complexes sediment (fractions 5-8) and used this pool as the INPUT for an anti-ORF1p co-immunoprecipitation (IP) followed by RNA-seq.
Project description:Unveiling the impact of carbon sources on phosphorus release from sediment: Investigation of microbial interactions and metabolic pathways for anaerobic phosphorus recovery