Quantifluor one dsdna kit

SMS libraries were prepared using the Nextera XT DNA Library Preparation Kit (# FC-131-1096, Illumina), following Illumina’s instructions (protocol # 15031942 v03 February 2018). Briefly, 1 ng of genomic DNA was used for the tagmentation reaction for a total volume of 20 µl. After 5 min at 55 °C, the reaction was stopped by adding 5 µl of the Neutralize Tagment (NT) Buffer. A limited-cycle PCR amplification was then performed to amplify the tagmentated DNA [addition of 15 μl of Nextera PCR Master Mix (NPM)] and to add Illumina sequencing adapters (addition of 5 µl of both Index 1 primer and Index 2 primer from the Nextera XT index kit, Illumina) for a total volume of 50 µl. The following PCR cycle program was used: 72 °C for 3 min, 95 °C for 30 s, 12 cycles of (95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min. SMS libraries were quantified using the QuantiFluor One dsDNA kit (# E4870, Promega) with the GloMax system (Promega). The quality of libraries was assessed using the High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. Sequencing was performed on a NextSeq500 system (Illumina) with the NextSeq 500/550 High Output v2 kit (300 cycles) in 2 × 150 bp.

The activity of ALP for OBs and TRAP, CAII and CTSK activity for OCs were measured and normalized to the DNA content of the respective samples. Frozen membranes with OBs, respectively well plates with PBMC/OCs were thawed and cells were lysed by incubation in 1 % Triton X-100 in PBS for 50 min with an ultrasonication step for 10 min in between. Quantification of osteoclastic TRAP, CAII and CTSK activities was performed as previously described [24 (link)]. Briefly, ALP and CAII activities were determined calorimetrically by the cleavage of colourless p-nitrophenyl phosphate (ALP) and p-nitrophenyl acetate (CAII) respectively into yellowish p-nitrophenol in different buffer solutions and quantified by absorbance measurements at 405 nm using a spectrofluorometer infinite M200pro (Tecan Trading AG, Switzerland). TRAP activity was measured by cleavage of naphthol ASBI phosphate at acidic pH in the presence of tartrate and detection of the emitted fluorescence signals at an excitation and emission wavelength of 405/520 nm. CTSK activity was quantified by the cleavage of Z-LR-AMC (Enzo Life sciences) with fluorescence measurements at an excitation and emission wavelength of 365/440 nm. DNA concentration of cell lysates was quantified using Quantifluor One dsDNA kit (Promega) according to manufacturer's instructions.

We collected buccal swap samples from 64 father-son pairs from European populations with written informed consent signed by all participants. Our analyses are based on self-declared family relations. DNA extraction was performed on the BioRobot® EZ1 (Qiagen, Hilden, Germany) using the EZ1 DNA Blood 200 µL Kit (Qiagen). Quantification of DNA samples was performed with the Rotor-Gene Q (Qiagen) and the Investigator Quantiplex Kit (Qiagen) or with the Quantus^™ Fluorometer (Promega Corporation, Madison, WI, USA) and the QuantiFluor^® ONE dsDNA Kit (Promega Corporation).

The V3-V4 region of bacterial 16S rDNA was PCR amplified with V3-340F (CCTACGGRAGGCAGCAG) and V4-805R (GGACTACHVGGGTWTCTAAT) barcoded primers. PCR products were cleaned using Ampure magnetic purification beads (Agencourt AMPure XP Kit), quantified with the QuantiFluor ONE dsDNA kit (Promega), and pooled in equal amounts of each PCR product. Library pools were loaded at 12 pM with a 15% PhiX spike for diversity and sequencing control onto a v3 300 bp paired end reads cartridge for sequencing on the Illumina MiSeq NGS platform. After removing reads containing incorrect primer or barcode sequences and sequences with more than one ambiguous base, a total of 2,905,540 reads (121,064 mapped reads on average) was recovered from the 24 studied samples. Bioinformatics analysis was performed as described in ref. 57 (link). All read sequences were deposited in the Sequence Read Archive (SRA) (submission: SUB8079771/BioProject ID: PRJNA661179).

Genomic DNA (gDNA) and total RNA were isolated from cell lines using TRIzol (Invitrogen) according to the manufacturer's protocol. gDNA was also isolated from formalin-fixed paraffin-embedded (FFPE) colorectal tissues with the AllPrep DNA/RNA FFPE Kit (Qiagen). cfDNA was isolated from 2–4 mL of plasma using the QIAamp® Circulating Nucleic Acid Kit (Qiagen) and the vacuum system QIAvac 24 Plus (Qiagen) following the manufacturer's recommendations. The quality and quantity of gDNA and RNA were evaluated with a NanoDrop (Thermo Fisher), and cfDNA was quantified by the QuantiFluor® ONE dsDNA kit with the Quantus™ Fluorometer (Promega). DNA and RNA were stored at − 80 °C until analysis.

16s ribosomal RNA library preparation, quantification, and sequencing were performed as recently published by Klymiuk et al.¹⁸ (link) Briefly, 5 μl of total DNA were used for PCR amplification with the target-specific primers 27F 5' - AGAGTTGATCCTGGCTCAG - 3' and R357 5' - CTGCTGCCTYCCGTA - 3' (MWG-Biotech AG, Germany ). The PCR reaction consisted of an initial denaturation at 95° C for 3 minutes, followed by 32 cycles of denaturation at 95° C for 45 seconds, annealing at 55° C for 45 seconds, and extension at 72° C for 1 minute. The final extension was at 72° C for 7 minutes, and PCR reactions were performed in triplicate. The triplicate reactions were pooled, and 5 μl were visualized on a 1% agarose gel to verify amplification success. The normalization, indexing, pooling of individual PCR products, and purification of the final library were performed as described in Klymiuk et al.¹⁸ (link) The library was quantified using the QuantiFluor ONE dsDNA kit (Promega) according to the manufacturer's instructions and visualized on an Agilent 2100 Bioanalyzer (Agilent Technologies) using a high-sensitivity DNA assay according to the manufacturer's instructions. The 6 pM library was sequenced on a MiSeqII desktop sequencer (Illumina) using version 3 chemistry for 600 cycles. For data analysis, 20% PhiX control DNA and FastQ files were used.

DNA samples from the 15 bacterial cultures were prepared for WGS, using the Nextera XT DNA library preparation kit (Illumina, California, USA) according to the manufacturer’s recommendations. The resulting libraries were checked for their quality using the High-sensitivity DNA chip using the Agilent 2100 Bioanalyzer (Waldbroon, Germany) and quantified using the QuantiFluor One dsDNA kit (Promega). Paired-end (2 × 300 bp) sequencing was performed on a MiSeq sequencer using the MiSeq v3 kit (600 cycles) (Illumina, California, USA).