Zr fecal dna miniprep kit

The MDMs were seeded at a density of 1 million cells on a glass coverslip and cultured in a six-well plate under the same conditions as described above. After MAP infection, the MDMs were washed with RPMI and then fixed using ice-cold 100% ethanol. The coverslips were then stained with an acid-fast staining using the commercial BD Auramine M kit protocol (BD Biosciences) and a DAPI 300 nM solution (Roche Diagnostics, Indianapolis, IN, USA) for 30 min. Coverslips were mounted on a microscope slide using Fluoroshield (Sigma-Aldrich, St. Louis, MO, USA) and examined using the Zeiss Axio Observer Z1 (Zeiss Canada, Toronto, Ontario, Canada). Analyses were performed using the ZEN 2 (blue edition) v2.0.0.0 software at the imaging platform of the Department of Biology of the Université de Sherbrooke (Quebec, Canada). All images were taken with the 63× objective and consisted of a merged image of the eGFP, DAPI, and differential interference contrast (DIC) channels. To confirm the absence of MAP in control samples from both JD(–) and JD(+) cows, DNA was extracted from adherent monocytes, MDM, and PBMC using ZR Fecal DNA MiniPrep kit (Zymo Research Corp., Irvine, CA, USA) and qPCR was performed using the VETMAX Gold MAP Detection Kit (Life Technology Inc., Burlington, Ontario, Canada) as described previously (36 (link)).

Cecal (0.1 gram) DNA was extracted using ZR Fecal DNA MiniPrep Kit (Zymo Research), quantified by NanoDrop (NanoDrop Technologies), and amplified using primers based on published sequences (Supplementary Table S4). A dissociation step was included to analyze the melting profile of amplified products. In parallel, qPCR was done using ten-fold serial diluted synthetic DNA fragments of bacterial gene with known concentrations. Bacterial DNA concentration was calculated using standard curves of diluted synthetic DNA fragment.
Pyrosequencing of Tagged 16S rRNA Gene Amplicons of cecal DNA was done based on published methods^{57 (link)}. The V4 region of 16S rRNA gene was amplified and sequenced using Illumina MiSeq.

Cecal and fecal samples were stored at -80 ${}^{\circ }\mathrm{C}$ until analysis. DNA was extracted from 100 mg of samples using ZR Fecal DNA MiniPrep Kit (Zymo Research), quantified by the NanoDrop (NanoDrop Technologies), and amplified with the primers as described previously^{48 (link)}. Illumina MiSeq sequencing of variable region 4 (V4) of 16S ribosomal RNA was run following the published methods^{49 (link),50 (link)}. Raw 16S rRNA high throughput sequencing data from each of the three sequencing runs was demultiplexed using sabre (https://github.com/najoshi/sabre). Demultiplexed reads were quality filtered and dereplicated in QIIME2 with the DADA2 plugin^{51 (link),52 (link)}. Ribosomal sequence variants (RSVs) with fewer than 10 total reads and RSVs in five or fewer samples were filtered from the DADA2 feature table and representative sequence files.

DNA was extracted from each of 283 stool specimens. For processing, samples were thawed at 4°C and, in aliquots of 0.15 g per tube, resuspended in 1 ml of 1× phosphate-buffered saline. Cell lysis was initiated with two enzymatic incubations, first, using 5 µl of lysozyme (10 mg ml⁻¹; Amresco, Solon, OH), 13 µl of mutanolysin (11.7 U µl⁻¹; Sigma-Aldrich), and 3 µl of lysostaphin (4.5 U µl⁻¹; Sigma-Aldrich) for an incubation of 30 min at 37°C and, second, using 10 µl proteinase K (20 mg ml⁻¹; Research Products International, Mt. Prospect, IL), 50 µl 10% SDS, and 2 µl RNase (10 mg ml⁻¹) for an incubation of 45 min at 56°C. After the enzyme treatments, cells were disrupted by bead beating in tubes with lysing matrix B (0.1-mm silica spheres; MP Biomedicals, Solon, OH), at 6 m s⁻¹ for 40 s at room temperature in a FastPrep-24 (MP Biomedicals). The resulting crude lysate was processed using the ZR fecal DNA miniprep kit (Zymo, Irvine, CA) according to the manufacturer’s recommendations. The samples were eluted with 100 µl of ultrapure water into separate tubes. DNA concentrations in the samples were measured using the Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Molecular Probes, Invitrogen, Carlsbad, CA).