Glass beads

DNA was extracted from each fecal sample using the modified protocol of the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Briefly, 1 ml InhibitEX buffer and glass beads (0.5-mm diameter, Qiagen) were added to a 200 mg fecal sample. The mixture was homogenized and mixed at 60 Hz for 1 min (twice) using a homogenizer. DNA was purified in accordance with the manufacturer's instructions.
The V3-V4 region of the bacterial 16S ribosomal RNA genes was amplified by PCR using barcoded primers 341F 5′-CCTACGGGRSGCAGCAG-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3′ (Wang and Qian, 2009 (link)). PCRs were performed in a volume of 30 μl containing 15 μl 2 × KAPA Library Amplification ReadyMix, 1 μl each primer (10 μM), 50 ng template DNA, and ddH₂O (Pereira et al., 2017 (link)).
Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions, and quantified using Qubit^®2.0 (Invitrogen, Waltham, MA USA). All quantified amplicons were pooled to equalize the concentrations for sequencing using the Illumina MiSeq PE250 (Illumina, Inc., San Diego, CA, USA). The paired-end reads (~250 bp in length) were overlapped at their 3′ ends for concatenation into original longer tags using PANDAseq (https://github.com/neufeld/pandaseq, version 2.9).

Fecal specimens were stored at −80°C until use. The DNA from the fecal samples was recovered using the QIAamp DNA Stool kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions with some modification (10 (link)). Two hundred milligrams of the fecal specimens was weighed in a 2 ml microcentrifuge tube containing 0.3 g glass beads, and 180 µl of ATL lysis buffer solution was added (Qiagen). The suspension was mixed using a vortex mixer followed by bead beating three times for 30 s at a setting of 4,200 rpm using a bead beater (Yasui Kikai, Tokyo, Japan). In addition, 20 µl of proteinase K was added to the mixture. After heating at 56°C for 30 min, the bacterial cells in the samples were treated by bead beating in the same manner. After destruction of bacterial cells, total DNA was collected using a Qiagen column and purified according to the manufacturer’s instructions. The DNA was extracted in 200 µl of AE buffer (Qiagen) with 400 µl of ethanol and 20 µl of 3 M sodium acetate and kept at −20°C for 14–16 h for ethanol precipitation. The precipitated DNA was collected by centrifugation for 20 min at 20,000 × g.

Approximately 5 × 10⁶ HUVECs stimulated with TNFα for 0, 10 or 30 min were digested (3 min at 37°C) with 750 units of micrococcal nuclease (MNase; Sigma-Aldrich, Seelze, Germany). Mononucleosomal DNA was isolated following separation on 1.3% agarose gels using glass beads (Qiagen, Hilden, Germany), and average fragment lengths determined using a 2100 Bioanalyzer (Agilent). Libraries were generated using the NEBNext DNA Library Prep Master Mix Kit (New England Biolabs, Ipswich, USA) and paired-end (2 × 50-bp) sequenced on a HiSeq2000 platform (Illumina, Essex, UK) to comparable depths (that is, 181, 185 and 187 million reads for 0, 10 and 30 min samples, respectively). Obtained reads were processed using the toolkits FastQC [44 ] and FASTX [45 ], mapped to hg19 using Bowtie [46 (link)].