Dnase max kit

A 10-mm intestinal piece of each mouse was collected at sacrifice and was kept at –80°C for storage. Tissue was disrupted and homogenized using RNase-free stainless steel beads in the Bullet Blender Strom 24 blender (Next Advance), RNA was extracted using TRIzol reagent (ThermoFisher Scientific) and DNase Max kit (QIAGEN). The expression levels of murine Il1β, Il6, Il17a, Tnf, and Tgfb were measured in SI cells by real-time PCR.

Two grams of soil were snap-frozen with liquid nitrogen 1 week after rewetting or thawing and stored at −80 °C. RNA was isolated using the RNA PowerSoil^® Total RNA Isolation Kit according to the instructions of the manufacturer (Qiagen, Vedbæk, Denmark). DNA was removed from the isolated RNA using the DNaseMax Kit (Qiagen) with an extension of the DNase reaction to 1 h to ensure that all DNA was removed. RNA was reverse transcribed using RevertAid RT Reverse Transcription Kit (Thermo Fischer Scientific, Roskilde, Denmark) with random hexamer primers. DNA was co-isolated using RNA PowerSoil^® DNA Elution Accessory Kit (Qiagen). The quantity of isolated RNA and DNA was determined on a Qubit Fluorometer (Thermo Fischer Scientific), and the quality was determined on a 1% agarose gel. DNA contamination of the isolated RNA was checked using qPCR (see later for conditions). Isolation of RNA and DNA failed for one sample (see Tables S1–S4). This sample was excluded from further analysis.

During pseudo-steady state, broth samples were taken from the enrichment culture, directly frozen in liquid nitrogen and subsequently stored at −80°C. Five hundred microliter samples were thawed on ice, pelleted by centrifugation (21,000 × g, 2 min, 4°C) and used for total RNA extraction with the RNeasy PowerMicrobiome Kit (Qiagen, Hilden, Germany), following the manufacturer’s instruction with the addition of phenol:chloroform:isoamy alcohol (25:25:1) and β-mercaptoethanol (10 μL mL^–1 final concentration). Cell lysis was with a FastPrep-24 bead beater (MP Biomedicals, Fisher Scientific, Hampton, VA, United States, four successive cycles of 40 s at 6.0 m s^–1, 2 min incubation on ice between cycles). Total RNA extracts were subjected to DNase treatment to remove DNA contaminants by using the DNase Max Kit (Qiagen, Hilden, Germany) and further cleaned up and concentrated with the Agencourt AMpure XP magnetic beads (Beckman Coulter, Brea, CA, United States) before rRNA depletion. Integrity and quality of purified total RNA were assessed on a Tapestation 2200 (Agilent, Santa Clara, CA, United States) with the Agilent RNA screen-tapes (Agilent, Santa Clara, CA, United States) and the concentration was measured using Qubit RNA HS Assay Kit (Thermo Scientific Fisher, Waltham, MA, United States).

Biomass pellets resulting from 40-mL cultures were resuspended by pipetting in 0.5 mL of TRIzol reagent (Thermo Fisher Scientific SARL, Ecublens, Switzerland) and incubated for 5 min at room temperature. A volume of 0.1 mL of chloroform was added and the mixture was vortexed vigorously for 15 s, incubated for 2 min and centrifuged at 10,000 × g for 15 min. The supernatant was carefully collected, mixed with an equal volume of 100% ethanol and purified using the Direct-zol RNA MiniPrep kit (Zymo Research, Lucerna-Chem AG, Luzern, Switzerland), following the manufacturer's instructions with the following modifications. The nucleic acids were eluted in 50 μL of water and a 5-μL aliquot was withdrawn to be used as DNA reference sample in quantitative PCR. The remaining elution was then supplemented with 5 μL of DNase buffer and 1 μL of DNase I enzyme (DNase Max Kit, Qiagen), and incubated for 30 min at 37°C. The DNase I enzyme was then removed with 5 μL of DNase Removal Resin according to the instructions. RNA samples were quantified using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and stored at −80°C until further use.

Total DNA was extracted from quarter membranes using the DNeasy^® PowerWater^® Kit (Qiagen) according to the manufacturer’s protocol with some modifications. After 10 min at 65°C, a mechanical cell lysis was carried out using the Precellys^® 24 bead beater (Bertin Technologies) and the following programme: 4 lysis cycles of 20 s at 5000 r.p.m., with 5 s of pause between each cycle. DNA was eluted in 50 µl of the elution solution provided in the kit, and samples were stored at 4°C until being tested by real‐time PCR.
Total RNA was extracted from quarter membranes using the RNeasy^® PowerWater^® Kit (Qiagen) according to the manufacturer’s protocol with some modifications. Mechanical cell lysis was carried out as described above for DNA extraction. Nucleic acid was eluted in 50 µl of the elution solution provided in the kit. Genomic DNA was removed using the DNase™ Max Kit (Qiagen) following the manufacturer’s recommendations, except that DNase reaction was performed at 37°C for 30 min instead of 20 min. The successful elimination of genomic DNA was checked by testing RNA extracts directly by real‐time PCR targeting Bonamia ostreae 18S rDNA (see below).
cDNAs were synthesized using the Invitrogen™ SuperScript™ III Reverse Transcriptase kit (Thermo Fisher Scientific), following the manufacturer’s instructions. cDNAs were stored at 4°C until being tested by real‐time PCR.

Total RNA was isolated using TRIzol reagent (ThermoFisher Scientific) and DNase Max kit (QIAGEN) according to the manufacturer’s directions. For all RNA samples, cDNA was synthesized with random hexamer primers (Roche) and AMV reverse transcriptase (Roche). The expression levels of murine Il17a, Tnf, and Tgfb were measured in whole BM cells by real-time PCR. Changes in relative gene expression between vehicle and cPTH groups or control and low Ca diet groups were calculated using the 2^–ΔΔCT method with normalization to 18S rRNA. The primers used are provided in Supplementary Table 1.

Flag leaves of barley genotypes were collected from all experimental variants at two time points (T1 and T2), and they were immediately frozen in liquid nitrogen and stored at −80°C until analysis. Total RNA (1–2 µg) was extracted in triplicates using the Qiagen (RRID : SCR_008539, http://www.qiagen.com, Hilden, Germany) RNeasy Plant Mini Kit according to the manufacturer’s instructions. Genomic DNA contamination was removed twice, i.e., on-column DNase digestion (RNase-Free DNase Set, Qiagen) and using the DNase Max Kit (Qiagen) during and after RNA isolation, respectively. Three flag leaves sampled from different plants of each genotype in a pot formed one replication, and three such replications were examined. RNA quantity, quality, and integrity were evaluated following the study by Mikołajczak et al. (2022) (link). The construction of a cDNA library (TruSeq stranded mRNA) and mRNA sequencing were performed by Macrogen Inc. (RRID : SCR_014454, http://www.macrogen.com, Seoul, Republic of Korea) using an Illumina NovaSeq6000 platform with a 150-bp paired-end configuration and the number of read pairs ranging from 18.3 to 40.9 M per sample.