Proteinase k

The BCC was determined by metabarcoding of the V3/V4 region of the 16S rRNA gene. Filters dedicated to bacterial diversity were immediately flash-frozen in liquid nitrogen and stored at −80°C until processing. Blank dry filters were sampled simultaneously and used as a contamination control. The two experiments were processed simultaneously. Filters were cut into pieces and DNA was extracted using the NucleoSpin Plant II Mini Kit (Macherey Nagel Ref. 740770.50) according to the manufacturer’s instructions, with an additional lysis step performed for 2 h at 56°C with 25 μL of proteinase K (20 mg mL^–1, Macherey Nagel Ref. 740506) and 100 μL of lysozyme (20 mg mL^–1, Sigma ref 4403-5g). Libraries were prepared and sequenced by Génome Québec using the 341F/785R primers (Klindworth et al., 2013 (link)). Samples were sequenced on an Illumina MySeq using 2 × 300 pb and V3 chemistry. Data were processed using the SAMBA pipeline (v3.0.1)² developed by the IFREMER bioinformatics team (SeBiMER). This resulted in 2,120 amplicon sequence variants (ASVs), which were then clustered using dbOTU3 (Olesen et al., 2017 (link)), resulting in 1,502 ASVs (29% clustering) that were assigned against the Silva v138 database (Quast et al., 2013 (link)).

Mice were sacrificed at the indicated ages by cervical dislocation. Ovaries were extracted and immediately placed in cryo-buffer containing 50% PBS, 25% ethylene glycol and 25% DMSO (Sigma–Aldrich, Austria) and stored at −80°C. For oocyte extraction, ovaries were placed into a drop of cryo-buffer and disrupted using scalpel and forceps. Oocytes were collected and remaining cumulus cells were removed mechanically by repeated careful suction through glass capillaries. Prepared oocytes were then washed in PBS before they were individually placed into compartments of 96-well PCR plates (Life Technologies, Austria) containing 10 μl of oocyte-lysis buffer (Lee et al., 2012 (link)) (50 mM Tris-HCl, [p.H 8.5], 1 mM EDTA, 0.5% tween-20 [Sigma–Aldrich, Austria] and 200 μg/ml Proteinase K [Macherey–Nagel, Germany]). Samples covered stages from primary oocytes of 3 day-old mice up to mature oocytes of 40 day-old mice. Samples were lysed at 55°C for 2 hr, and incubated at 95°C for 10 min to inactivate Proteinase K. The cellular DNA extract was finally diluted in 190 μl Tris-EDTA buffer, pH 8.0 (Sigma–Aldrich, Austria). 3 μl were used per qPCR reaction.

As previously described [29 (link)], the reaction, including a PCR product (100 nM) (PCR protocol described in Section 2.4) and a ribonucleoprotein (RNP) complex consisting of sgRNAs (200 nM) and Cas9 nuclease (100 nM) (IDT, Coralville, IA, USA), was incubated at 37 °C for 2 h. The reaction was stopped by adding proteinase K (Macherey Nagel, Düren, Germany) for 10 min at 56 °C. Then, 1% agarose (Lonza, Basel, Switzerland) gel electrophoresis was used to visualize the resulting products.

Mice were killed at the indicated ages by cervical dislocation. Ovaries were extracted and immediately placed in cryo-buffer containing 50% PBS, 25% ethylene glycol and 25% DMSO (Sigma-Aldrich, Austria) and stored at −80 °C.
For oocyte extraction, ovaries were placed into a drop of cryo-buffer and disrupted using scalpel and forceps. Oocytes were collected and remaining cumulus cells were removed mechanically by repeated careful suction through glass capillaries. Naked oocytes were then washed in PBS before they were individually placed into compartments of 96-well PCR plates (Life Technologies, Austria) containing 10 μl oocyte-lysis buffer^{28 (link)} composed of 50 mM Tris-HCl, (pH 8.5), 1 mM EDTA, 0.5% Tween-20 (Sigma-Aldrich) and 200 μg/ml Proteinase-K (Macherey-Nagel). Samples covered stages from primary oocytes of 3-day-old mice up to major oocytes of adult mice. Samples were lysed at 55 °C for 2 h, and incubated at 95 °C for 10 min to inactivate Proteinase K. The cellular DNA was finally diluted in 190 μl Tris-EDTA buffer, pH 8.0 (Sigma-Aldrich).

Microdissected samples were kept overnight in lysis buffer containing proteinase K (0.2 U per sample) obtained from Macherey-Nagel (Düren, Germany). Samples were processed for specific PCR amplification the next day. To avoid cross contamination, blank DNA samples (water) were processed in parallel with the tissue samples. We measured DNA concentration using Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Grand Island, NY) with the Qubit DNA HS assay kit. Moreover, to evaluate the quality of DNA we used the TapeStation (Agilent Technologies, Santa Clara, CA) with Genomic DNA Screen Tape.