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11 protocols using magna lyser tube

1

Total RNA Isolation and S1 Nuclease Mapping

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For total RNA isolation, tissue was first grinded to powder cooled in liquid nitrogen. The frozen powder was transferred to a MagNA Lyser tube (Roche Applied Science, Indianapolis, IN) filled with 1 ml TRIzol (Invitrogen, Carlsbad, CA) and homogenized immediately by MagNA Lyser homogenizer (Roche Applied Science, Indianapolis, IN). The supernatant was used in RNA isolation following manufacturer’s instructions. For S1 nuclease mapping, appropriate restriction enzyme-digested DNA fragment was labeled at the 5’-end with [γ-32P] ATP and T4 polynucleotide kinase [11 ]. The labeled DNA probe was hybridized to total RNAs prepared from the mouse tissues. The DNA-RNA hybrids that resisted to S1 nuclease digestion were electrophoresed through a 4% polyacrylamide gel containing 7 M urea. Gel was dried and analyzed either by autoradiography or by a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
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2

Total RNA Extraction and Northern Blot

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To isolate total RNA, tissues were grinded to powder in liquid nitrogen. The frozen powder was transferred to TRIzol reagent (Invitrogen, Carlsbad, CA) in a MagNA Lyser tube (Roche Applied Science, Indianapolis, IN) and was homogenized. Supernatant was obtained for RNA isolation following manufacturer’s instructions. For northern blot analysis, RNA was denatured in glyoxal before electrophoresis on a 1.2% agarose gel. RNA was transferred to Hybond N+ nylon membrane (GE Healthcare Life Sciences, UK), hybridized to an EGFP or an ASS cDNA probe and analyzed by a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
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3

Viral and Bacterial RNA Extraction

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The faecal samples underwent a bead-beating pre-treatment carried out by adding faecal material to a solution of 900 μl of L6 lysis buffer (ThermoFisher Scientific) and 30 μl of isoamyl alcohol (Sigma-Aldrich). The suspension was placed in a MagNa Lyser tube (Roche) and shaken for 1 minute at 3000 rotations per minute in a MagNa Lyser vortex (Roche). After shredding, 300 μl of supernatant were mixed to 300 μl of PBS, vortexed, spun down and inserted into a QIAsymphony SP automatic extractor (QIAgen). During the extraction process, the samples were spiked with 102 plaque forming units of Bacteriophage MS2 and 105 colony forming units of Bacillus thuringiensis in AVE buffer (QIAgen) as external controls of extraction. The nucleic acids were purified using the DSP virus/pathogen mini kit (QIAgen), eluted in 110 μl of AVE buffer (QIAgen) and stored at –20°C until TAC analysis.
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4

RNA Analysis of Organoid Cultures

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For RNA analysis, three organoids per cell line were collected at days 52 and 110. The organoids were washed with PBS and frozen at-20oC. For RNA isolation the ReliaPrep RNA cell miniprep system (Promega) was used according to manufacturer’s instructions with a modification in the cell lysis step. For the cell lysis, lysis buffer was added to each tube containing an organoid and triturated to disperse the cell pellet/organoid. The cell lysate was transferred to a MagNA Lyser tube (Roche) for homogenization in a bullet blender at intensity 8 for 3 min (or 3 × 1 min). After homogenization the lysates were centrifuged for 3 min at 10.000 × g and the supernatant was transferred into a new tube to continue with the protocol according to manufacturer’s instructions. RNA concentration was measured by Qubit Fluorometer (Invitrogen) and RNA samples were stored at-80oC. For cDNA synthesis 100 ng of total RNA from each organoid was used as template in the Transcriptor first strand cDNA synthesis kit with random hexamer primers (Roche). We validated our primer sets with RT-qPCR using a LightCycler 480 Real-Time PCR System (Roche) with SsoFast EvaGreen Supermix with low ROX (BioRad). The cycling parameters of the qPCR included an initial denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s. CT-values were normalized to GAPDH using the ΔΔCT-method.
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5

Novodiag Stool Parasites Detection Assay

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Sample preparation and Novodiag® Stool Parasites assay (Hologic formerly Mobidiag, Espoo, Finland) was performed according to the manufacturer’s instructions. In brief, the stool was collected with a sterile swab (eSwab tube, Copan, Brescia, Italy) from several places in the faeces. The swab was then placed in the eSwab tube and vortexed for 5 s. In case of liquid stool, 200 μL was taken and then directly added into the eSwab tube.
The entire contents of the eSwab tube were then transferred into a MagNALyser tube (Roche Diagnostics, Mannheim, Germany). After bead beating at 7000 rpm for 90 s (MagNALyser, Roche Diagnostics, Mannheim, Germany), 600 μL of the lysate was then transferred into the NSP cartridge using a sterile single-use pipette under a class II biological safety cabinet (laminar flow). The cartridge was inserted into the Novodiag instrument, and the results (Positive/Negative/Invalid) were automatically displayed on the instrument screen at the end of the reaction.
According to the manufacturer’s instructions, every sample with an invalid result was retested a second time with a new test tube (new eSwab tube from the original sample) and, if needed, a third time with a diluted sample (300 μL of the first eSwab tube transferred into a second eSwab tube).
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6

High-Yield Total RNA Extraction from IVLE

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IVLE frozen in Trizol reagent were subjected to 3 freeze-thaw cycles by manually transferring tubes between a water bath at 95°C and dry ice. This procedure enhanced the total RNA yield from the samples. Thereafter, the eggs were transferred to MagNA Lyser tubes (Roche) containing ceramic beads, homogenized in FastPrep (FastPrep-24, MP Biomedicals) at setting 6 with two 20-second pulses and incubated for 5 minutes at room temperature. To each sample, 150μl chloroform was added, shaken vigorously for 10 seconds, incubated for 3 minutes at room temperature and centrifuged at 15,000g for 15 minutes at room temperature. The aqueous phase was carefully removed to a clean centrifuge tube, and the RNA was precipitated by adding one volume of 100% ethanol and incubating at -80°C overnight. The samples were centrifuged at maximum speed for 30 minutes at 4°C, the RNA pellet washed in 70% ethanol, air dried and resuspended in nuclease free water. The RNA quality was assessed and quantified using the Bioanalyzer (2100 Bioanalyzer Instrument, Agilent Technologies). Although the yield of RNA was modest, <30 ng total, high-quality RNA was recovered from each replicate sample (Supplementary Figure S1A).
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7

Colon Tissue RNA Extraction for Sequencing

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Distal colon tissues were collected in Magnalyser tubes (Roche, Basel, Switzerland) and ground in 700 µL of Qiazol lysis buffer (Qiagen, Hilden, Germany) with the FastPrep-24 5G (MPBio, Irvine, CA, USA). Total RNAs (including microRNAs) were purified using miRNeasy Mini Kit (Catalog number 217004, Qiagen) according to the manufacturer’s instructions.
RNA concentration and purity were determined using a UV spectrophotometer (NanoDrop ND1000, Thermo Fisher Scientific) by measuring the absorbance at 230, 260, and 280 nm. The integrity of the RNA was further checked with the Agilent 2100 Bioanalyzer, and RNAs with RIN values > 7 were used in the downstream RNAseq workflow.
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8

Bovine Fecal DNA Extraction Protocol

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A DNA extraction method described in detail previously was used to isolate genomic DNA from bovine feces [13 (link),14 (link)]. Briefly, approximately 500 to 700 mg from each fecal specimen were resuspended in 2.0 ml of 0.15 M phosphate buffered saline (PBS, 136.9 mM NaCl, 1.46 mM KH2PO4, 8.1 mM Na2HPO4 × 2H2O, 2.7 mM KCl, pH 7.4). 100 μL of the sample suspensions were mixed with 130 μL lysis buffer and 20 μL of Proteinase K provided in the MaGNA Pure DNA Isolation Kit III (Roche Molecular Diagnostics, Penzberg, Germany) and incubated for 30 min at 65°C and 10 min at 95°C. The mixtures were transferred onto a bead-beating matrix in MagNA lyser tubes (Roche). A mechanical lysis step consisting of 60 s at 6,500 rpm followed by 60 s on a cooling block held at 4°C was performed three times on the samples. 100 μL of sample lysates were transferred to the MagNA Pure LC instrument sample cartridge. DNA was automatically isolated from these samples using the MagNA Pure LC instrument and in accordance with the protocol of the MagNA Pure LC DNA Isolation Kit III. Purified DNA templates were eluted in 100 μL of elution buffer provided in the DNA isolation kit. A 2 μL aliquot from the final eluate was used as a template for PCR.
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9

RNA Extraction and qRT-PCR Analysis

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Pancreatic tissue was homogenized using MagNa Lyser tubes (Roche, catalog 03358941001) in TRIzol reagent (Thermo Fisher Scientific, catalog 15596026) and RNA was isolated using Qiagen RNEasy Plus Mini kit (Qiagen, catalog 74134) according to manufacturer protocol. cDNA was isolated using high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, catalog 4368814). The primer sequences used are provided in the Table 1. Quantitative real-time detection of cDNA was performed using Light Cycler 480 II (Roche) after adding the primer mixes and SYBR green (LightCycler 480 SYBR Green I Master, catalog 04887352001) in triplicate according to the manufacturer recommendations.
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10

High-Yield Total RNA Extraction from IVLE

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IVLE frozen in Trizol reagent were subjected to 3 freeze-thaw cycles by manually transferring tubes between a water bath at 95°C and dry ice. This procedure enhanced the total RNA yield from the samples. Thereafter, the eggs were transferred to MagNA Lyser tubes (Roche) containing ceramic beads, homogenized in FastPrep (FastPrep-24, MP Biomedicals) at setting 6 with two 20-second pulses and incubated for 5 minutes at room temperature. To each sample, 150μl chloroform was added, shaken vigorously for 10 seconds, incubated for 3 minutes at room temperature and centrifuged at 15,000g for 15 minutes at room temperature. The aqueous phase was carefully removed to a clean centrifuge tube, and the RNA was precipitated by adding one volume of 100% ethanol and incubating at -80°C overnight. The samples were centrifuged at maximum speed for 30 minutes at 4°C, the RNA pellet washed in 70% ethanol, air dried and resuspended in nuclease free water. The RNA quality was assessed and quantified using the Bioanalyzer (2100 Bioanalyzer Instrument, Agilent Technologies). Although the yield of RNA was modest, <30 ng total, high-quality RNA was recovered from each replicate sample (Supplementary Figure S1A).
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