Tris edta

Six healthy volunteers including a 4-year-old child and 5 adults (32 ± 3 years old) were recruited from BGI Europe employees or family members, Copenhagen, Denmark (see detailed information in Supplementary Table 2). All volunteers or the guardian provided informed consent to provide fecal samples for this study. Roughly 10–15 grams of stool were freshly collected by participants at home by using a 50-mL sterile conical tube, and copies of printed instructions were used to guide the adult volunteers or the child's legal guardian for self-collection of fecal samples. After collection, samples were stored at −20°C and transported to the laboratory on the second day with ice packs in 40 minutes. Then, each sample was diluted with 1–1.5 volumes (15 mL) of Tris-EDTA (10 mM Tris pH 8.0 and 1 mM EDTA, Thermo Fisher Scientific) buffer, homogenized, and divided into 36 aliquots (500 µL per aliquot). All stool aliquots were stored at −80°C before DNA extraction.

To isolate total RNA, the Qiagen RNeasy minikit was used following the RNAprotect bacterial reagent handbook, with modifications. P. aeruginosa biofilms were grown as described above. From the MBEC plate, ∼180 μl of medium was collected from each well and placed into 1.5-ml centrifuge tubes. Collected medium was then centrifuged for 2 min at 12,000 rpm. Pelleted cells were resuspended in 200 μl of RNase free water. Each tube was then centrifuged again for 2 min at 12,000 rpm. The remaining supernatant was discarded. After centrifugation, 30 μl of a 10 mg/ml solution of lysozyme (MP Biomedicals) in 1× Tris-EDTA (Thermo Fisher) was added to each centrifuge tube, followed by incubation at room temperature for 20 min with vortexing every 2 min. The total RNA was then extracted according to the manufacturer’s recommended protocol, including the optional in-column DNase digestion using the RNase-Free DNase set. After RNA extraction, we used a Qiagen DNase Max kit according to the Quick Start protocol provided by the manufacturer. cDNA synthesis was performed using Bio-Rad reverse transcription supermix according to the manufacturer’s recommendations in a Bio-Rad C1000 touch thermal cycler.

Unstained and paraffin-embedded ovarian tissue slides were deparaffinized with xylene and rehydrated with graded ethanol. For antigen retrieval, the slides were treated with Tris-EDTA (pH 9.0; Thermo Scientific) for 30 min at room temperature. The samples were blocked with hydrogen peroxide solution (Cat#: 88597, Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The slides were stained using a Polink-2 Plus HRP Broad Kit (Cat#: D41-18, GBI Labs, Bothell, WA, USA) with DAB (3,3′3diaminobenzidine), according to the manufacturer’s protocol. The slides were incubated at 4 °C overnight in a humid chamber with primary antibodies: γ-H2AX (1:500 dilution; Bethyl Laboratories, Montgomery, AL, USA), 8-OHdG (1:500, Cat#: sc-66036, Santa Cruz, Dallas, TX, USA), PARP (1:400, Cat#: 46D11, Cell Signaling Technology, Danvers, MA, USA), and CDKN2A/p16INK4a (1:100, Cat#: EPR20418, Abcam, Cambridge, MA, USA) in PBS containing 2% BSA. The slides were incubated for 1 h with the following secondary antibodies: goat anti-rabbit IgG (H + L), Alexa Fluor 488 conjugate (1:1000, Invitrogen, Waltham, MA, USA), goat anti-rabbit IgG (H + L), and Alexa Fluor 594 conjugate (1:1000, Invitrogen). The slides were counterstained with Mayer’s hematoxylin (Cat#: HMM125, Scytek, West Logan, WV, USA) and antifade mounting medium with DAPI (Cat#: H-1200, Vector Laboratories, Burlingame, CA, USA).