Frozen samples of liver and gill tissue were processed together as a single extraction for each sample. The combined tissues were placed in 600 μl of lysis buffer containing 0.5 per cent foaming reagent (Reagent DX, Qiagen) and 1 per cent of β-mercaptoethanol (Sigma-Aldrich). Submerged tissue samples were homogenized with TissueRuptor (Qiagen) for one minute at 5,000 rpm. To further homogenize tissue samples and remove tissue residues, the homogenate was centrifuged at full speed for three minutes. The homogenate was carefully removed and RNA from the clear supernatant was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Extracted RNA was quantified using NanoDrop (ThermoFisher) and RNA from each species was pooled corresponding to the site in which they were captured, resulting in thirty-six sample libraries (Supplementary Table S2 ). RNA libraries were constructed using the Truseq Total RNA Library Preparation Protocol (Illumina). To enhance viral discovery and reduce the presence of non-viral reads, host ribosomal RNA (rRNA) was depleted using the Ribo-Zero-Gold Kit (Illumina) and paired-end sequencing (150 bp) was performed on the NovaSeq 500 platform (Illlumina). Sample library construction, rRNA depletion and RNA sequencing were performed at the Australian Genome Research Facility.
Reagent dx
Manufactured by Qiagen
Sourced in United Kingdom, Germany, France
Reagent DX is a laboratory reagent product developed by Qiagen. It is designed to facilitate various analytical and diagnostic procedures in a laboratory setting. The core function of Reagent DX is to provide a consistent and reliable solution for the required chemical reactions and processes involved in these laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Rneasy plus mini kit, by Qiagen (4 mentions)
Truseq total rna library preparation protocol, by Illumina (2 mentions)
Ribo zero gold kit, by Illumina (2 mentions)
Tissueruptor, by Qiagen (2 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Lysis buffer, by Qiagen (2 mentions)
70 micron smartstrainer, by Miltenyi Biotec (2 mentions)
30 μm smartstrainer, by Miltenyi Biotec (2 mentions)
Sytox blue live dead stain, by Thermo Fisher Scientific (2 mentions)
2 mercaptoenthanol, by Merck Group (2 mentions)
Facsaria 2, by BD (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Atl buffer, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Pathogen lysis tube, by Qiagen (1 mentions)
Nanodrop uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Bead ruptor elite bead mill homogenizer, by Omni International (1 mentions)
Tissuelyser system, by Qiagen (1 mentions)
19 protocols using reagent dx
1
Comprehensive RNA Extraction and Sequencing
2
Cell Wall Lysis via Bead Beating and DNA Extraction
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Mechanical lysis of cell walls was accomplished with bead beating. One aliquot of enriched urine samples (200 μl) was transferred into Pathogen Lysis Tubes (Qiagen) with glass beads, and 50 μl of Buffer ATL (containing Reagent DX, Qiagen) was added according to the manufacturer’s instructions. The Pathogen Lysis Tubes were then attached to a horizontal platform on a vortex mixer and vortexed for 10 min at maximum speed. After that, the Pathogen Lysis Tubes were removed and briefly spined to collect any drops from the inside of the lid. DNA was extracted from the supernatant using the IndiSpin Pathogen Kit as described in Method 1.
3
Cortical Tissue RNA Extraction
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Brains were homogenized using the Bead Ruptor Elite Bead Mill Homogenizer (#19-042E, OMNI International) in homogenization buffer from the RNeasy Plus Micro Kit (#74034, Qiagen) with Reagent DX added at 1:200 dilution (#1011008, Qiagen) to reduce foaming. Total RNA was extracted from frozen cortical tissue using RNeasy Plus Micro Kit following the manufacturer’s instructions. RNA quality and concentration were measured using a NanoDrop UV–vis spectrophotometer (#13-400-519, ThermoFisher).
4
Salmonella DNA Isolation Protocol
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Salmonella DNA was isolated in a QIAcube HT robot using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, Valencia, CA). A single Salmonella colony was suspended into 5 ml of TSB and incubated overnight at 37 °C. From the suspension culture, 1 ml was transferred into a 1.2 ml micro-collection tube and centrifuged at 4,000 rpm for 15 minutes at room temperature. After the supernatant was removed, the pellet was re-suspended in ATL buffer (Qiagen) mixed with reagent DX (Qiagen). One tube of small pathogen lysis beads (Qiagen) was mixed with the suspension, and disrupted with the Qiagen TissueLyser system (Qiagen) at 25 Hz, for 5 minutes. The tubes were briefly centrifuged and 40 μl of Proteinase K was added to each tube. The tubes were incubated at 56 °C for 1 hour at 900 rpm in a ThermoMixer (Eppendorf, Hauppauge, NY) followed by a heat shock for 10 minutes at 95 °C. The suspension was cooled to room temperature and 4 μl of RNAse A was added. The prepared samples were set in the QIAcube HT for DNA extraction using a modified protocol provided by Qiagen. The quality of the DNA was determined by the 260/280 ratios on the FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC). The DNA quantity was measured with a Quant-iT™ Pico Green® ds DNA Assay kit (Thermo Fisher Scientific) and the DNA was stored at −20 °C until future use.
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Salmonella DNA was isolated in a QIAcube HT robot using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, Valencia, CA). A single Salmonella colony was suspended into 5 ml of TSB and incubated overnight at 37 °C. From the suspension culture, 1 ml was transferred into a 1.2 ml micro-collection tube and centrifuged at 4,000 rpm for 15 minutes at room temperature. After the supernatant was removed, the pellet was re-suspended in ATL buffer (Qiagen) mixed with reagent DX (Qiagen). One tube of small pathogen lysis beads (Qiagen) was mixed with the suspension, and disrupted with the Qiagen TissueLyser system (Qiagen) at 25 Hz, for 5 minutes. The tubes were briefly centrifuged and 40 μl of Proteinase K was added to each tube. The tubes were incubated at 56 °C for 1 hour at 900 rpm in a ThermoMixer (Eppendorf, Hauppauge, NY) followed by a heat shock for 10 minutes at 95 °C. The suspension was cooled to room temperature and 4 μl of RNAse A was added. The prepared samples were set in the QIAcube HT for DNA extraction using a modified protocol provided by Qiagen. The quality of the DNA was determined by the 260/280 ratios on the FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC). The DNA quantity was measured with a Quant-iT™ Pico Green® ds DNA Assay kit (Thermo Fisher Scientific) and the DNA was stored at −20 °C until future use.
5
Quantitative RT-PCR Gene Expression Analysis
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Prior to lysis, cells were washed twice with PBS. Lysis buffer buffer RLT
(Qiagen, Manchester, UK) supplemented with 0.05% Reagent DX (Qiagen) was added
to each well and cells lysed using pipette tip. RNA was extracted using AllPrep
DNA/RNA/miRNA universal kit (Qiagen), following manufacturers’ protocol. Equal
amounts of RNA were used for complementary DNA (cDNA) synthesis using Taqman
Reverse Transcription Reagents (Thermo Fisher Scientific) according to the
manufacturers’ protocol. Amplification by quantitative reverse
transcriptase-polymerase chain reaction (qRT-PCR) was performed using
human-specific primers (Sigma). Each 20-μL reaction contained 1 μL of diluted
cDNA, 10 μL of GoTaq (Promega, Southampton, UK) and a final concentration of
1 μM forward and reverse primers. For the qRT-PCR run, Applied Biosystems
(Thermo Fisher Scientific) Real-Time PCR system was set up to run first with two
holding stages (2 min at 50°C, 10 min 95°C), followed by 40 cycles (15 s at
95°C, 1 min at 60°C) where fluorescence was measured and held at 4°C. Data were
analysed using 7500 Software version 2.3 (Life Technologies, Basingstoke, UK).
Threshold for calculating Ct (cycle threshold) value was set at 0.2 and all
samples normalised to expression of ACTB as a housekeeping
gene. The 2−ΔΔCt method was used for relative quantification of gene expression.27 (link)
(Qiagen, Manchester, UK) supplemented with 0.05% Reagent DX (Qiagen) was added
to each well and cells lysed using pipette tip. RNA was extracted using AllPrep
DNA/RNA/miRNA universal kit (Qiagen), following manufacturers’ protocol. Equal
amounts of RNA were used for complementary DNA (cDNA) synthesis using Taqman
Reverse Transcription Reagents (Thermo Fisher Scientific) according to the
manufacturers’ protocol. Amplification by quantitative reverse
transcriptase-polymerase chain reaction (qRT-PCR) was performed using
human-specific primers (Sigma). Each 20-μL reaction contained 1 μL of diluted
cDNA, 10 μL of GoTaq (Promega, Southampton, UK) and a final concentration of
1 μM forward and reverse primers. For the qRT-PCR run, Applied Biosystems
(Thermo Fisher Scientific) Real-Time PCR system was set up to run first with two
holding stages (2 min at 50°C, 10 min 95°C), followed by 40 cycles (15 s at
95°C, 1 min at 60°C) where fluorescence was measured and held at 4°C. Data were
analysed using 7500 Software version 2.3 (Life Technologies, Basingstoke, UK).
Threshold for calculating Ct (cycle threshold) value was set at 0.2 and all
samples normalised to expression of ACTB as a housekeeping
gene. The 2−ΔΔCt method was used for relative quantification of gene expression.27 (link)
6
Single-Cell Isolation of CX3CR1+ Cells
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Brain tissues were collected from perfused male MgWT and MgPTX mice as previously described41 (link). Single-cell suspensions were prepared from tissue containing cortical and hippocampal regions following the adult brain dissociation (ABD) kit manufacturer’s instructions with modification (Miltenyi Biotec). Briefly, minced tissues were incubated with ABD Mix 1 for 15 min at 34 °C, and then ABD Mix 2 was added for 10 min at 34 °C. Tissues were gently triturated and then incubated for 10 min at 34°C. Homogenized tissue solutions were passed through 70-μm smartstrainer (Miltenyi Biotec), washed with cold Dulbecco’s PBS and centrifuged at 450xg for 7 min at 4°C. Tissue debris was removed following the ABD Kit debris removal step, followed by straining through 30-μm smartstrainer (Miltenyi Biotec) and then centrifuged at 450xg for 7 min at 4°C. Single-cell suspensions were incubated with 1 μM Sytox blue live/dead stain (Thermo Fisher Scientific) for 5 min at 4°C and then cell sorting was performed on an FACSARIA II (BD Biosciences). Live Sytox blue− CX3CR1-GFP+ cells were sorted directly into tubes containing RLT plus lysis buffer (Qiagen) supplemented with 1% 2-mercaptoenthanol (Sigma) and 0.25% reagent DX (Qiagen). Cell lysates were frozen on dry ice and immediately stored at −80°C until use.
7
Automated Skin Biopsy Lysis with Customizable Homogenization
8
RNA Extraction from Snap-Frozen Tissue
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Snap frozen material was disrupted using TissueLyser LT (Qiagen), 5 mm stainless steel beads (Qiagen) and reagent DX (Qiagen). The lysate was further homogenized using QIAshredder (Qiagen) and RNA was extracted using RNeasy mini kit (Qiagen) with on column DNAse digest (Qiagen) and additional washing steps. The quality of the RNA was assessed using an Experion RNA StdSens Analysis Kit (Bio-Rad).
9
Rothia and Moraxella Infection of Airway Epithelium
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We centrifuged liquid cultures of R. aeria strain RSM41, R. dentocariosa strain RSM522, and R. similmucilaginosa strain RSM42 at 5,000 × g for 5 min and resuspended the cell pellet to an OD600 of ~0.25 in prewarmed PBS. We cultured M. catarrhalis on BD Chocolate Agar–GC II Agar with IsoVitaleX and prepared a suspension of the bacterial cells to an OD600 of ~0.25 in prewarmed PBS. We inoculated the apical surface of separate wells of ALI cultures with 50 μL of a Rothia suspension for 40 h. We infected ALI cells with M. catarrhalis as previously described (98 (link)). Briefly, we infected the apical surface with 50 μL of the M. catarrhalis suspension and incubated the cells at 37°C for 24 h. At the end of the infection period, we gently washed the apical surface with 500 μL prewarmed PBS and collected the cells in 350 μL of RLT Plus Buffer (Qiagen) containing 0.5% Reagent DX (Qiagen).
10
RNA Isolation and qRT-PCR from Cultured Cells
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Cultured c-Kit+ or LSK-SLAM cells were centrifuged, lysed in 350 μl RLT Plus Lysis buffer with Reagent DX (Qiagen) and beta-mercaptoethanol (Sigma-Aldrich), and RNA was isolated using the RNeasy Plus Micro kit (Qiagen) as indicated by the manufacturer. For spike-in confirming successful RNA isolation from cultured cells, 150 ng YFP mRNA was added at the time of lysis. RNA concentration was quantified on a NanoDrop 1000 (ThermoFisher). 50 ng total RNA per sample was used for first-strand cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen) as indicated by the manufacturer. Relative gene expression was quantified on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems). Expression levels of genes of interest were normalized to β-actin expression. RT-PCR primers were designed using NCBI Primer-BLAST, and sequences are available upon request.
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