List of proteins detected by mass spectrometry in endogenous Strep MIWI complexes.

MIWI elution fractions were analysed by mass spectrometry at the EMBL Proteomics Core Facility (Heidelberg) and results are featured in this table. To isolate endogenous MIWI complexes, the MiwiStrep knock-in line (RP86) and the Strep-Tactin-XT Sepharose chromatography resin (Cytiva, Cat. No. 29401326) that interacts with the StrepII tag. All extractions were carried out in the cold room at 4°C. The resin was packed into Poly-Prep Chromatography Columns (Biorad). For every purification, we used 1 ml of resin slurry and this gave a bed-volume equivalent to 450 mL in these columns. Columns were washed 3 times with water, and then with Streptactin wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA). Five pairs of testes from adult (>P60, 60 days after birth) homozygous the MiwiStrep animals were lysed by douncing in a modified lysis buffer (10% glycerol, 50 mM Tris/HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5% Na deoxycholate, 1% TritonX 100, 1x proteinase inhibitors, and 10 mM Vanadyl complex), supplemented with BioLock (IBA Lifesciences, Cat. No. 2-0205-050) to reduce the binding of other endogenous biotinylated proteins to the resin. Previous purifications were performed without BioLock in this lysis step. We used 0.75 mL of lysis buffer per testis. The lysate was incubated on ice for 30 min in a douncer. The extract was cleared by two centrifugations steps at 9,200 g, 4°C, for 15 min, then was added to the resin and incubated for 2 h at 4°C. The column was sealed at both ends and then placed on a rotating wheel to mix. After collection of the flow-through, the column was further washed 5 times with Streptactin wash buffer (5 x 8 mL). Washing conditions were unchanged for purifications with and without BioLock. The bound protein was eluted with a total of 1.5 mL Streptactin elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 50 mM Biotin), and the eluate was collected in ~0.2-0.25 mL fractions. In total, 6 consecutive fractions were collected.