Bullet blender storm tissue homogenizer

Tissue from each organ (30 mg) was individually placed into a sterile Red RINO lysis tube (Next Advance, Troy, NY) containing 600 μL of Buffer RLT and 6 μL β-mercaptoethanol. Samples were mechanically disrupted using the Bullet Blender Storm Tissue Homogenizer (Next Advance) for 3 min at speed level 12. Samples were processed thereafter using the RNeasy Mini Kit (QIAGEN) following the RNeasy Kit protocol, including the optional on-column DNase digestion using DNaseI (Thermo Fischer Scientific, AM2222; Waltham, MA). RNA concentrations were determined using the Experion Automated Electrophoresis System and Experion RNA StdSens chips (Bio-Rad Laboratories, Hercules, CA). Only RNA samples with an RNA quality indicator (RQI) value of ≥8 (1–10 scale of quality) were used for transcript identification. RNA samples were stored at −80°C until further analysis.

Bacterial DNA was extracted according to a published protocol (21 (link)). Previously frozen fecal samples (30 mg) were placed in 800 μL of lysis buffer (500 mM NaCl, 50 mM tris-HCl, 50 mM EDTA, and 4% SDS), homogenized for 5 min at speed level 12 using the Bullet Blender Storm Tissue Homogenizer (Next Advance), and incubated at 70°C for 20 min. Samples underwent centrifugation at 5,000 g for 5 min at room temperature. The supernatant was mixed with 200 μL of 10 mM ammonium acetate, incubated on ice for 5 min, and centrifuged at 16,000 g for 10 min at room temperature. An aliquot of supernatant (750 μL) was mixed with equal volumes of chilled isopropanol and incubated for 30 min on ice. Samples were centrifuged at 14,000 g and 4°C for 15 min to pellet DNA. Pellets were rinsed twice with 70% ethanol and re-suspended in 150 μL Tris-EDTA. Proteinase K (15 μL) and buffer AL (200 μL; DNeasy Kit, QIAGEN) were added and samples were incubated at 70°C for 10 min. Then 100% ethanol (200 μL) was added and samples were transferred to a QIAGEN DNeasy spin column and processed per the manufacturer’s instructions for DNA purification (DNeasy Kit, QIAGEN). Samples were eluted in 100 μL EB buffer (QIAGEN) and dsDNA yield was measured using Qubit dsDNA BR assay kits and fluorometry (Qubit 2.0, Life Technologies, Carlsbad, CA).