Qubit fluorescence assay

Each water sample was aseptically filtered through a polyethersulfone membrane (0.22 μm pore size, 50 mm diameter) using vacuum-driven filters (Biofil) and a vacuum-pressure pump (Millipore). Filter membranes were cut into small pieces using a stainless steel scalpel and placed in sterile tubes. A control using ultrapure water was carried out to verify whether the membrane or filtration process could introduce any contamination. Bacterial genomic DNA was extracted from bacterial cells retained on each filter membrane using the DNeasy PowerSoil Kit (Qiagen), following the manufacturer’s methods. A reagent blank extraction was the control of the DNA extraction process. DNA sample concentration was measured using a Qubit fluorescence assay (Invitrogen). All DNA samples were concentrated in a vacufuge concentrator (Eppendorf) and sent for amplicon sequencing (16S rRNA, V4 region primers) on an Illumina HiSeq PE250 instrument at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Since the controls, both from the filtration process and the DNA extraction resulted in negative PCR amplification, they were not further processed and were not sequenced (Additional file 2).

Whole cell proteomic analysis was performed in MSCs at different passages (P3, P5, and P7) by the LC/MS proteomic platform. MSCs were cultured at different passages, and cell pellets were collected and washed in ice-cold PBS (pH 7.2) followed by treatment with urea lysis buffer (8 M urea in 0.1 M Tris-HCl, pH 8.5). Protein estimation was performed by Qubit fluorescence assay (Invitrogen). A total of 50 μg protein was digested using the FASP procedure as described previously [15 ]. Liquid chromatography tandem mass spectrometric analysis of tryptic peptides (500 ng) was carried out using a Proxeon nano spray ESI source (Thermo Fisher, Hemel, UK) and analyzed using Orbitrap Velos Pro FTMS (Thermo Finnigan, Bremen, Germany) [16 (link)].

Total RNA was extracted from cells using RNeasy Plus mini kits (Qiagen). RNA concentration was measured by a Qubit fluorescence assay (Thermo Fisher, United States). A total 1 μg of extracted RNA was reverse transcribed using SuperScript IV VILO Master Mix kit (Thermo Fisher, United States), according to manufacturer’s instructions. cDNA was diluted 1:10 in nuclease free water prior to qPCR analysis using a CFX384 real-time PCR system (Bio-Rad, United States). Reaction mixtures containing 5 μL PowerUp SYBR Green Master Mix (Applied Biosystems, United States), 4 μL cDNA, 1 μL primer mix (final concentration of 200 nM per primer) were prepared in Hard-Shell PCR 384-well plates (Bio-Rad, United States). Amplification conditions were as follows: 50°C for 2 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. Melting curve analysis was performed from 65°C–95°C. All samples were run in triplicate and mean Ct (cycle threshold) values were used for further analysis.