E gel

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E-gel is a pre-cast agarose gel designed for DNA analysis and separation. It provides a convenient and consistent platform for running electrophoresis experiments.

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109 protocols using e gel

Sequencing Library Preparation from Gregaria DNA

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The concentration of the
S. gregaria high molecular weight DNA sample was measured with PicoGreen (Invitrogen) fluorimetry, after which DNA integrity was confirmed by gel electrophoresis (1% E-Gel; Invitrogen). The sample was divided for Illumina MP and PE sequencing library preparation.
The MP sequencing library was prepared from 1 µg of the sample with a “Nextera Mate Pair Library prep kit” (Illumina). The PE library was prepared with a “NEBNext Ultra II library prep kit” (NEB) from 2 µg of the sample, sheared to 500 bp fragments using an S2 focused-ultrasonicator (Covaris). Size selection (600–700 bp) was performed for both libraries in a 2% E-Gel (Invitrogen). The quality of the libraries was confirmed with a Bioanalyzer High Sensitivity DNA Kit (Agilent). The MP and PE libraries were quantified by qPCR, according to Illumina's “Sequencing Library qPCR Quantification protocol guide” (version February 2011) and pooled at a molar ratio of 25% MP – 75% PE for sequencing on Hiseq3000 (2 × 150 cycles, 16 lanes; Illumina).

Verlinden H., Sterck L., Li J., Li Z., Yssel A., Gansemans Y., Verdonck R., Holtof M., Song H., Behmer S.T., Sword G.A., Matheson T., Ott S.R., Deforce D., Van Nieuwerburgh F., Van de Peer Y, & Vanden Broeck J. (2021). First draft genome assembly of the desert locust, Schistocerca gregaria. F1000Research, 9, 775.

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Chitosan–siRNA Nanoparticle Stability Assay

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Stability
of chitosan–siRNA nanoparticles against RNase digestion was
investigated. Chitosan–siRNA nanoparticles in an amount equivalent
to 2 μg of siRNA were tested. The nanoparticles were prepared
as previously described and in a N/P ratio of 50/1. Each set of particles
was in phosphate buffer at pH 7.4 and was subjected to four different
conditions: no treatment; incubated for 10 min with 5 μL of
heparin (1000 U/mL) for displacing the siRNA from the chitosan nanoparticles;
incubated with 4 μL of RNase A (20ug/mL) for 30 min at 37 °C;
and incubation with incubation 4 μL of RNase A (20ug/mL) for
30 min at 37 °C followed by heat inactivation of the enzyme and
incubation for 10 min with 5 μL of heparin (1000 U/mL). Resulting
mixtures were applied to a 4% E-Gel (Invitrogen, Carlsbad, CA), and
electrophoresis was carried for 15 min using the E-Gel iBase system.
In each gel, free siRNA was applied as a reference. The experiments
were performed in triplicate. The resulting gel was imaged on ChemiDoc
System (Bio Rad, Waltham, MA) using the software Quantity One.

Nascimento A.V., Singh A., Bousbaa H., Ferreira D., Sarmento B, & Amiji M.M. (2014). Mad2 Checkpoint Gene Silencing Using Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles in Non-Small Cell Lung Cancer Model. Molecular Pharmaceutics, 11(10), 3515-3527.

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Isolation and Characterization of Diaphragm Muscle RNA

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Diaphragm muscle samples were removed from storage at −80 °C, weighed and samples ranging from 20–50 mg were homogenized on ice in Tripure Isolation Reagent (Roche Diagnostics, Ltd., Burgess Hill, UK) using a general laboratory homogenizer (Omni-Inc., Kennesaw, GA, USA). Total RNA was isolated from homogenates in accordance with the manufacturer’s instructions, with an additional chloroform wash step performed during phase separation. The quantity (ng μL⁻¹) and purity of isolated RNA (260:280 & 260:230 ratios) was assessed using a Nanodrop 1000 (Thermo Scientific, Waltham, MA, USA) and spectrophotometry. The integrity of isolated RNA was assessed by visualization of distinct 18S and 28S ribosomal RNA bands using an agarose gel electrophoresis system (E-gel, Life Technologies, Carlsbad, CA, USA).

Burns D.P., Drummond S.E., Bolger D., Coiscaud A., Murphy K.H., Edge D, & O’Halloran K.D. (2019). N-acetylcysteine Decreases Fibrosis and Increases Force-Generating Capacity of mdx Diaphragm. Antioxidants, 8(12), 581.

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Fecal Microbiome DNA Extraction and Sequencing

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Microbial DNA was extracted from fecal samples using a PureLink Microbiome DNA purification kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), according to the manufacturer’s instructions following a preliminary step of bead-beating for 2 min and elution in 50 μl elution buffer. Purified DNA was subjected to PCR amplification using PrimeSTAR Max (TaKaRa-Clontech, Shiga, Japan) for the variable V4 region (using 515F-806R barcoded primers) of the 16S rRNA gene, as previously described (22 (link)).
Amplicons were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA) and subsequently quantified using a Quant-It PicoGreen double-stranded DNA (dsDNA) quantitation kit (Invitrogen, Carlsbad, CA). Equimolar amounts of DNA from individual samples were pooled, cleaned by the use of E-gel (Life Technologies, Carlsbad, CA, USA), and sequenced using the Illumina MiSeq platform at the Genomic Center of the Bar-Ilan University, at the Azrieli Faculty of Medicine.

Lapidot Y., Amir A., Nosenko R., Uzan-Yulzari A., Veitsman E., Cohen-Ezra O., Davidov Y., Weiss P., Bradichevski T., Segev S., Koren O., Safran M, & Ben-Ari Z. (2020). Alterations in the Gut Microbiome in the Progression of Cirrhosis to Hepatocellular Carcinoma. mSystems, 5(3), e00153-20.

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Diaphragm Muscle RNA Extraction

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Diaphragm muscle samples from each experimental group (sham, CIH, CIH + APO, NOX2 KO sham, and NOX2 KO CIH; n = 6–9 per group) were examined. Samples were weighed following removal from storage at −80 °C. A general lab homogeniser (Omni-Inc., Kennesaw, GA, USA) was used to homogenise samples (20–40 mg) in Tripure Isolating reagent (Roche Diagnostics Ltd., West Sussex, UK) kept on ice. The resultant homogenates were placed on ice for 20 min with intermittent vortexing to promote cell lysis. The phenol-chloroform method was utilised to extract total RNA from homogenates, in accordance with the manufacturer’s instructions. For muscle samples, an additional chloroform wash step was performed to enhance the purity of isolated RNA. By spectrophotometry, a nanodrop 1000 (Thermo Scientific, Waltham, MA, USA) was used to assess both the quantity (ng/µL) and purity of isolated RNA (260:280 and 260:230 ratios). Using an agarose gel electrophoresis system (E-gel, Life Technologies, Carlsbad, CA, USA), the integrity of isolated RNA was assessed by visualization of distinct 18S and 28S ribosomal RNA bands.

Drummond S.E., Burns D.P., El Maghrani S., Ziegler O., Healy V, & O’Halloran K.D. (2023). Chronic Intermittent Hypoxia-Induced Diaphragm Muscle Weakness Is NADPH Oxidase-2 Dependent. Cells, 12(14), 1834.

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qPCR Analysis of Gene Expression

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cDNAs obtained from RT of RNA were diluted 10-fold and 4 μL were mixed with SYBR®Green Master Mix (Biorad, Nazareth, Belgium) to a final volume of 20 μL containing 300nM of each primer. Amplification was carried out in the CFX96 thermal cycler (BioRad) under the following conditions: heating for 3 min at 95°C, 40 cycles of denaturation for 30 s at 95°C, followed by an annealing/extension for 1 min. After each run a melting curve analysis was performed, ramping from 55°C to 95°C in 20 min. A negative control without cDNA template was run in every assay and measures were performed in duplicates.
Primers were designed with the Beacon Designer Pro 8.10 software (Premier Biosoft, Palo Alto, USA), flanking an intron. Specificity was assessed using the NCBI BLAST tool [71 ], melting curves were performed in each assay and gene-specific amplification was confirmed by a single band in 4% E-Gel® (Life technologies). Data analysis normalization was carried out against three reference genes; ef1a, rpl13a, tuba1 and expression levels were calculated using the CFX manager 3.0 software (Biorad) via the delta-delta Cq method, taking into account the calculated amplification efficiency for each primers pair. See Additional file 5 for MIQE checklist [72 (link)] and details of qPCR experiments.

Rodius S., Nazarov P.V., Nepomuceno-Chamorro I.A., Jeanty C., González-Rosa J.M., Ibberson M., da Costa R.M., Xenarios I., Mercader N, & Azuaje F. (2014). Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models. BMC Genomics, 15(1), 852.

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Brd2, Brd4, H3K27ac, H3K4me1 ChIP-seq Protocol

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ChIPed-DNA was end repaired with T4 DNA polymerase and polynucleotide kinase. An A-base was added to the end-repaired DNA fragments. Solexa adaptors were ligated to the DNA fragments and 200–300bp size fractions were obtained using E-gel (Life Technologies). Adaptor-modified fragments were enriched by 18 cycles of PCR amplification. The DNA library prep was validated in Bioanalyzer for quantity and size. The input- and ChIPed-DNA libraries were sequenced on the Illumina HiSeq2000 platform with 50bp read length in a single end mode. Brd2 and Brd4 ChIP-seq were performed and analyzed in triplicate. H3K27ac and H3K4me1 ChIP-seq were performed in duplicate. Smc1, Smc3 and Nipbl were performed once. All ChIP-seq data described in this study have been deposited in GEO under the accession number GSE90788 and GSE63778.

Cheung K.L., Zhang F., Jaganathan A., Sharma R., Zhang Q., Konuma T., Shen T., Lee J.Y., Ren C., Chen C.H., Lu G., Olson M.R., Zhang W., Kaplan M.H., Littman D.R., Walsh M.J., Xiong H., Zeng L, & Zhou M.M. (2017). Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation. Molecular cell, 65(6), 1068-1080.e5.

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Illumina-based 16S rRNA sequencing

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Samples were amplified for sequencing variable regions 1 to 2 (V1‐V2) at RTL Genomics (Lubbock, TX) in process consisting of two steps. The forward primer was constructed with the Illumina i5 sequencing. The reverse primer was constructed with the Illumina i7 sequencing primer. The laboratory performed amplifications with HotStar Taq Master Mix (Qiagen Inc) in reactions on ABI Veriti thermal cycler (Applied Biosytems, Carlsbad, CA). First stage amplification products were added to a second polymerase chain reaction (Nextera PCR primers; Illumina, Inc, San Diego, CA) based on qualitatively determined concentrations. EGel (Life Technologies, Grand Island, NY) were used to visualize amplification products. They were pooled equimolar and selected in two rounds using SPRIselect (Beckman Coulter, Indianapolis, IN). Size selected pools were then run on a Fragment Analyzer (Advanced Analytical, Ankeny, IA) to assess the size distribution, quantified using the Qubit 2.0 fluorometer (Life Technologies), and loaded on an Illumina MiSeq (Illumina, Inc) and sequenced. Sequence data were processed for denoising and chimera checking using a research and testing pipeline that is described together with details about used primers in http://www.researchandtesting.com/docs/Data_Analysis_Methodology.pdf.

Veit‐Rubin N., De Tayrac R., Cartwright R., Franklin‐Revill L., Warembourg S., Dunyach‐Remy C., Lavigne J, & Khullar V. (2019). Abnormal vaginal microbiome associated with vaginal mesh complications. Neurourology and Urodynamics, 38(8), 2255-2263.

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Metagenomic Library Preparation and Sequencing

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Metagenomic libraries were prepared from isolated DNA using the NEB DNA Mastermix Library preparation kit (New England Biolabs), with size selection by eGel (Life Technologies). Libraries were sequenced for 250-bp paired-end reads using the Illumina MiSeq reagent kit V2 on an Illumina MiSeq sequencer. After quality filtering with Trimmomatic (39 (link)), the paired and unpaired forward reads were combined and reads with more than 10% ambiguous bases were removed. Next, the reads were screened for human sequences with Best Match Tagger v3.101 (K. Rotmistrovky and R. Agarwala, 2010), after which human reads and duplicate reads were removed.

Zaura E., Brandt B.W., Teixeira de Mattos M.J., Buijs M.J., Caspers M.P., Rashid M.U., Weintraub A., Nord C.E., Savell A., Hu Y., Coates A.R., Hubank M., Spratt D.A., Wilson M., Keijser B.J, & Crielaard W. (2015). Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces. mBio, 6(6), e01693-15.

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Mitochondrial DNA Library Preparation

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The mitochondrial DNA libraries were generated using a protocol adapted from Wilkening et al. (2013) (link). One to 10 µg of mitochondrial DNA was sheared using the S220 Focused ultrasonicator (Covaris) with the following conditions: duty cycle, 20%; intensity, 5; cycles per burst, 200; duration, 45 sec; temperature, 4°. The 350-bp fragments were purified using 1 vol Agencourt AMPure XP beads (Beckman Coulter). The fragments were submitted to end repair, A addition, and adapter ligation with enzyme heat inactivation after each step. The samples were purified using 1 vol Agencourt AMPure XP beads and the libraries were amplified by PCR (98° for 45 sec; 12 cycles of 98° for 15 sec, 65° for 30 sec, and 72° for 30 sec; and 72° for 5 min). This step was performed using 2–10 ng of DNA, 2× Phusion High Fidelity PCR Master Mix (New England Biolabs, Beverly, MA), and 0.2 µM PE1.0 and PE2.0 primers (Illumina, San Diego, CA). The PCR products were then cleaned with 1 vol Agencourt AMPure XP beads and the yield was quantified with a Qubit Fluorometer (Life Technologies, Carlsbad, CA). Up to 13 samples were pooled and purified for 400- to 450-bp fragments on an E-gel (Life Technologies, Carlsbad, CA). The multiplexed libraries were sequenced on a Hiseq 2000, yielding between 2.8 and 42 million reads per sample (Table S2).

Fritsch E.S., Chabbert C.D., Klaus B, & Steinmetz L.M. (2014). A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast. Genetics, 198(2), 755-771.

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