Feces were collected on the first day of the first and on the last day of the third DSS cycle. Samples were snap frozen in liquid nitrogen and stored at −20 °C. DNA was isolated using the QIAamp Fast DNA stool Mini kit (Qiagen) according to the manufacturer’s instructions, with bead beating using Lysing Matrix E tubes (MP Biomedicals) before extraction. 16S rRNA gene amplicon sequencing and library preparation was performed using a standard Illumina protocol [36 ]. Reads were processed using the software packages DADA2 [37 (link)] and SINA [38 (link)]. For the analysis of sample similarity modified Rhea scripts were used [39 (link)]. Generalized UniFrac distances were visualized using multi-dimensional scaling [40 (link)]. We assessed cluster significance using permutational multivariate analysis of variance. Testing for significant differences in diversity and bacterial abundances was performed using Kruskal-Wallis Rank Sum Test with Benjamin-Hochberg method for correction for multiple comparisons. We used the Mann-Whitney U test to compare phylogenetic distances.
Lysing matrix e tube
Manufactured by MP Biomedicals
Sourced in United States, Germany, France, Australia
Lysing Matrix E tubes are designed for efficient mechanical disruption of biological samples. The tubes contain a proprietary blend of beads that help to lyse cells and release their contents during the homogenization process. The tubes are suitable for use with a variety of sample types, including microbial cultures, plant tissues, and animal tissues.
Automatically generated - may contain errors
Lab products found in correlation
Fastprep 24, by MP Biomedicals (15 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (9 mentions)
Proteinase k, by Qiagen (7 mentions)
Rlt lysis buffer, by Qiagen (7 mentions)
Allprep dna rna protein kit, by Qiagen (7 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (7 mentions)
Tissuelyser, by Qiagen (6 mentions)
Fastprep 24 instrument, by MP Biomedicals (6 mentions)
Dneasy blood and tissue kit, by Qiagen (6 mentions)
Allprep dna rna mini kit, by Qiagen (6 mentions)
Tissuelyser 2, by Qiagen (5 mentions)
Fastdna spin kit for soil, by MP Biomedicals (5 mentions)
Atl buffer, by Qiagen (4 mentions)
Ez1 dna tissue kit, by Qiagen (4 mentions)
Ez1 advanced xl instrument, by Qiagen (4 mentions)
Rlt buffer, by Qiagen (4 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Fastprep 24 homogenizer, by MP Biomedicals (3 mentions)
Floqswab, by Copan (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
89 protocols using lysing matrix e tube
1
Microbiome Analysis in Murine Colitis
2
Extraction of DNA for qPCR and Cloning
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DNA for quantitative PCR (qPCR) and cloning PCR was extracted from cells collected from 1 to 2 mL culture by centrifugation at 4°C and 13,400 rpm for 30 min. DNA was extracted using a modified version of the protocol by Griffiths et al. (2000 (link)) and Urich et al. (2008 (link)), as follows: cells were resuspended in pre-warmed (65°C) 1% sodium dodecyl sulfate (SDS) extraction buffer and transferred to Lysing Matrix E tubes (MP Biomedicals) containing an equal volume of phenol/chloroform/isoamylalcohol (25:24:1). Cell lysis was performed in a FastPrep-24 (MP) device with speed setting 4 for 30 s, and the lysate was centrifuged at 13,400 rpm for 10 min. An equal volume of chloroform/isoamylalcohol (24:1) was added to the supernatant of the lysate, followed by centrifugation at 13,400 rpm for 10 min and collection of the aqueous phase. Nucleic acids were precipitated with an equal volume of isopropanol with addition of 40 μL NaCl 5 M and 1 μL glycogen (20 mg mL−1) as carrier, incubated for 1 h at room temperature. Following centrifugation at 13,400 rpm for 1 h, nucleic acid pellets were washed with 1 mL cold 70% ethanol, dried at 30°C using a SpeedVac centrifuge, eluted in nuclease-free water and stored at −20°C until analysis. Nucleic acid quantification for metagenome sequencing was performed with a Qubit™ 2.0 Fluorometer (Invitrogen) using the dsDNA HS Assay Kit.
3
Gut Microbiome DNA Extraction
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The collected gut samples were added to Lysing Matrix E tubes containing ceramic beads (MP Biochemicals GmbH, Eschwege, Germany). After adding PBS (400 μl) to the samples, they were homogenised with a Precellys 24 Tissue Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Nucleic acid was extracted using the FastDNA Spin Kit for Soil (MP Biochemicals GmbH, Eschwege, Germany) following the manufacturer’s instructions.
4
DNA Extraction from Filters and Sediments
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DNA was extracted as described previously for filters (Orsi et al., 2015 (link)) and sediments (Coskun et al., 2018 (link)). DNA from the sediments was performed exactly as described previously (Coskun et al., 2018 (link)). For the filters, some modifications to the original DNA extraction protocol (Orsi et al., 2015 (link)) were made. For the filters, the silica bead contents from four 2 mL Lysing Matrix E tubes (MP Biomedicals, Solon, OH, USA) was poured into a 15-ml falcon tube containing the filter 4 ml of a sterile-filtered sucrose ethylene-diaminetetraacetic lysis buffer (0.75 M sucrose, 0.05 M Tris-Base, 0.02 M ethylenediaminetetraacetic, 0.4 M NaCl, 4 ml 10% sodium dodecyl sulfate, and pH 9.0) was added to the tubes and then bead beating was performed for 40 s using a Fast-Prep 5G homogenizer (MP Biomedicals, OH, USA) at a speed of 6 m/s. Samples were subsequently heated for 2 min at 99°C. After heating, 25 ml of 20 mg/ml proteinase K was added, and tubes were incubated at 55°C overnight with constant gentle rolling in a Bambino oven. DNA was extracted and purified from the lysate using the DNeasy Blood and Tissue Kit (Qiagen). Extracted DNA was quantified fluorometrically using a Qubit 3.0 Fluorometer (Invitrogen, Eugene, OR, USA).
5
Fecal DNA Isolation from Live Mice
6
Microbial DNA Extraction from Anaerobic Consortium
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Microbial DNA was extracted from the anaerobic consortium as described previously (74 (link)), with modifications. Briefly, an aliquot of a biological replicate from the ERAC culture was centrifuged at 12,000 × g for 20 min at 4°C. The resulting pellet was suspended in lysis buffer (100 mM EDTA, 50 mM NaCl, 10 mM Tris, pH 8, 1% SDS, proteinase K). The mixture was incubated at 37°C for 1 h with shaking. To ensure cell lysis, a bead-beating step was carried out using Lysing Matrix E tubes (MP Biomedicals), followed by incubation in a water bath at 65°C for 2 h. After centrifugation, the supernatant was mixed with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol). The solution was centrifuged, and the aqueous phase was transferred to a clean tube and treated with RNase A (Qiagen, Germantown, MD, USA) for 15 min at 37°C. The DNA was precipitated with isopropanol and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA solution was purified using Power Clean DNA clean-up kits (Mo Bio Laboratories) for the following applications.
7
Optimized CTAB DNA Extraction from Stool
8
Transcriptomic Analysis of Arabidopsis Immune Response
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Plants were germinated with the indicated SynCom in the presence or absence of 1 µM flg22 and incubated for 14 d before harvesting. For transcriptome experiments, Col-0 plants were inoculated with SynCom NS3 and SynCom S3 (Fig. 3 ); pWER::FLS2-GFP plants were inoculated with SynCom NS1 and SynCom S1 (Extended Data Fig. 6 ). For transcriptome experiments, plants were not transferred to minimize induced damage. Roots from three plates (minimum of 15 plants) were combined as one replicate and a total of three replicates were sampled for each condition. Roots were washed briefly with sterile water and blotted dry before being transferred into Lysing Matrix E tubes (MP Biomedicals) at −80 °C until processing. Roots were homogenized with Lysing Matrix E using a prechilled adapter and the TissueLyser II (QIAGEN, 20 pulses per s for 1 min). RNA was extracted using the Plant RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. RNA quality was determined using a 2100 Bioanalyzer (Agilent Technologies). Preparation of Illumina sequencing libraries was conducted by the Max Planck Genome Center using an input of 1 μg total RNA. Sequences were generated using the Illumina HiSeq2500 platform. Approximately 6 million paired-end reads and 20 million single-end reads per sample with a length of 150 bp were generated for Col-0- and pWER::FLS2-GFP-based experiments.
9
Rectal Swab DNA Extraction Protocol
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Rectal swabs (FLOQSwabs; Copan Diagnostics, Murrieta, CA) were collected under direct mucosal visualization via anoscopy without preparatory enema then immediately frozen neat at −80°C until processing in bulk. Samples were lysed by transferring to Lysing Matrix E tubes (MP Biomedicals, Burlingame, CA) containing RLT lysis buffer (QIAGEN, Hilden, Germany) and bead-beated on a TissueLyser (QIAGEN). Deoxyribonucleic acid (DNA) was then extracted using the AllPrep DNA/RNA/Protein kit (QIAGEN) per the manufacturer’s protocol.
10
Osedax root tissue DNA extraction
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DNA was extracted from 10- to 50-mg samples of either scraped biofilm or Osedax root tissue, using the Qiagen AllPrep DNA/RNA minikit according to the manufacturer’s instructions with cell lysis by a bead beating step in Lysing Matrix E tubes (MP Biomedicals) in a FastPrep homogenizer (MP Biomedicals) with a single cycle of 30 s at a speed of 5,500 rpm. The obtained DNA was quantified and quality controlled using a NanoDrop2000 (ThermoFisher Scientific) and a Qubit fluorometer 3.0 (ThermoFisher Scientific). The obtained DNA concentrations ranged from 11.9 ng/μl to 166 ng/μl according to Qubit readings. The DNA libraries were prepared using the Nextera DNA library prep kit according to the manufacturer’s guidelines. Fifty nanograms of DNA was used for the preparations. In brief, the DNA was fragmented by the Nextera transposome at 55°C for 5 min and barcoded adapters were added in a 5-cycle PCR amplification. The resulting libraries, including ∼140 bp of adapter, had an average fragment size of 436 bp (±112 bp) and were sequenced on an Illumina HiSeq4000 platform (150-bp paired-end reads) at the Institute of Clinical Molecular Biology (IKMB), Kiel University, Germany.
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