Protease k

According to a suggested method in the previous study, the thawed ileal digesta were digested with 1 mg/mL Protease K (Sigma, St Louis, MO, USA) in a buffer containing 50 mmol/L Tris (pH 7.5), 100 mmol/L EDTA, and 0.5% SDS for 5 h at 55 °C. Ileal microbiota DNA was then isolated by phenol–chloroform extraction following ethanol precipitation [29 (link)]. The concentration of extracted DNA was determined by a Nano-Drop spectrophotometer (Thermo, Wilmington, DE, USA). The V4-V5 region of the bacteria 16S rRNA gene was amplified by polymerase chain reaction (PCR) using primers 515F (5’-barcode- GTGCCAGCMGCCGCGG-3’) and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’), of which the reactions conditions were consistent with previous reports [30 (link)]. PCR products were extracted from 2% (w/v) agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions. Then, Purified PCR products were quantified by Qubit®3.0 (Invitrogen, Carlsbad, CA, USA) and used to construct the pair-end library following Illumina’s genomic DNA library preparation procedure [31 (link)]. The raw reads were deposited into the Sequence Read Archive database under the accession number: SRP365994.

Mouse platelets were prepared from 5 mL mixed blood collected from six Hpa-tg and Ctr mice. Human platelets were from mixed blood donated by 3 healthy individuals. After washing with PBS, platelets were lysed in 50 mMTris-HCl/0.5 M NaCl/1% Triton X-100, pH 7.4. Lysates were subjected to protease K (Sigma) digestion (0.8 mg/mL protease in 50 mMTris-HCl/1mM CaCl₂/1% Triton X-100, pH7.4) for 24 hr at 55°C. The homogenates were boiled (5 min) to inactivate the protease. After centrifugation at 13,000×g for 10 min, the supernatants were recovered and applied onto a 1-mL DEAE-Sephacel column (GE Healthcare Biosciences) pre-equilibrated with 50 mM Tris-HCl/0.1M NaCl, pH7.4. The column was washed with 50 mMNaAc/0.1 M NaCl, pH 4.5 and proteoglycans (PGs) were eluted with 50 mMNaAc/2M NaCl, pH 4.5. The eluted fractions were pooled and desalted on a PD-10 column (GE Healthcare Biosciences), followed by lyophilization to dryness. Aliquots of the samples were treated with buffer, Chondroitinase ABC (0.1 U/sample, Seikagaku, Japan) or Heparinase I/Heparinase III (1 mU/sample, Seikagaku, Japan) for 24 hr at 37°C. After heat inactivation of the enzymes, samples were separated on 15% PAGE and visualized by Alcian blue 8GX (Sigma-Aldrich) staining.

The ChIP assays were performed by using EZ-ChIP KIT according to the manufacturer’s instruction (Millipore, USA). The control and KDM5B siRNA-treated Hep3B and Focus cells were treated with formaldehyde and incubated for 10 min to generate DNA-protein cross-links. Then the cell lysates were sonicated to generate chromatin fragments of 200–1000 bp. The antibodies against KDM5B and trimethylated H3-K4 (H3-K4me3) were used to precipitate DNA fragments bound by their corresponding elements. The protein-DNA complex was collected with protein A Sepharose beads (Millipore, USA), eluted, and reverse cross-linked. Following treatment with Protease K (Sigma-Aldrich, USA), the samples were extracted with phenol-chloroform and precipitated with ethanol. The recovered DNA was resuspended in TE buffer and used for the PCR amplification.