Total RNA was extracted from mouse lungs using the TRIzol® reagent (Thermo Fisher Scientific) according to the instructions of the manufacturer (n = 4–5, each group). The concentration and purity of the RNA samples were determined by automated optical density evaluation (OD260/OD280 ≥ 1.8 and OD260/OD230 ≥ 1.8) using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA sequencing (RNA-seq) libraries were prepared using a NEBNext rRNA Depletion Kit (New England Biolabs, Ipswich, MA, USA) and an ENBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) according to the instructions of the manufacturer using 500 ng of the total RNA samples. Next, 2 × 36 base paired-end sequencing was performed using a NextSeq 500 sequencer (Illumina, San Diego, CA, USA) by Tsukuba i-Laboratory LLP (Tsukuba, Japan). Sequences were mapped to the mm10 mouse genome and quantified using CLC Genomics Workbench version 10.1.1 (QIAGEN). An adjusted P-value <0.01 (Benjamini–Hochberg FDR method for multiple testing corrections) and relative changes in transcription levels >1.2-fold were used as the cutoff criterion (Supplemental Figure 1
Clc genomics workbench version 10
Manufactured by Qiagen
Sourced in Denmark
CLC Genomics Workbench version 10.1.1 is a bioinformatics software platform designed for the analysis and visualization of genomic data. It provides a comprehensive suite of tools for tasks such as sequence assembly, annotation, and comparative analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext rrna depletion kit, by New England Biolabs (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Enbnext ultra directional rna library prep kit, by New England Biolabs (1 mentions)
R version 4, by R Project for Statistical Computing (1 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Nextflex rapid dna sequencing kit, by PSC Biotech (1 mentions)
Wizard dna kit, by Promega (1 mentions)
Nextera xt dna preparation kit, by Illumina (1 mentions)
Statview ver5, by SAS Institute (1 mentions)
12 protocols using clc genomics workbench version 10
1
RNA-seq Analysis of Mouse Lung Transcriptome
2
Differential Gene Expression Analysis
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Sequencing reads were mapped on the hg19 reference genome, quantified, and then normalized via quantile method using CLC Genomics Workbench version 10.1.1 (Qiagen, Aarhus, Denmark). DEG analyses were performed using the Empirical Analysis of DGE tools in CLC Genomics Workbench. The statistical threshold for DEGs was defined as a false discovery rate (FDR) < 0.1 or raw p value < 0.05, and absolute fold-change > 2.0. Principal component analysis (PCA) plots, heatmaps, and volcano plots were generated using R (version 4.0.2; R Foundation, Vienna, Austria).
3
RNA-seq Analysis of JQ1-Treated 46G-F Cells
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Total RNA of 46G-F cells with or without JQ1-treatment was extracted using ISOGEN plus. Thereafter, 500 ng of total RNA were ribosomal RNA-depleted using a NEBNext rRNA Depletion Kit (New England Biolabs). The resulting total RNA was converted to an Illumina sequencing library using a NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). The library was validated to determine the size distribution and concentration using a bioanalyzer (Agilent Technologies). Sequencing was performed on NextSeq 500 (Illumina) with paired-end 36-base read options. Reads were mapped to the hg19 human reference genome and quantified using CLC Genomics Workbench version 10.1.1 (Qiagen). RNA-seq data sets have been deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (NCBI) with the accession number GSE140476.
4
Variant Calling and Clustering Analysis of RNA-seq Data
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Variant calling was performed using the Basic Variant Call tool in CLC Genomics Workbench version 10.1.1 (Qiagen) to obtain a list of SNP positions and allele frequencies of variants in the RNA-seq reads. The B6 reference read was defined as a read aligned to the reference genome (mm10). The Sp variant read was defined as a read not assigned to the reference genome. To distinguish sequence errors from true polymorphisms, only variants found in more than two samples were classified as polymorphisms between B6 and Sp. At each polymorphism, B6 and Sp allele frequencies were calculated as B6 reference read number/(B6 reference read number + Sp variant read number) and Sp variant read number/(B6 reference read number + Sp variant read number), respectively. B6 gene frequency was calculated as an average of B6 allele frequencies of all polymorphisms in the gene. For clustering according to the B6 gene frequency profile, we used Ward’s agglomerative hierarchical clustering on the basis of a Euclidean distance matrix computed using the standard R functions hclust() with method = “ward.D2” and dist() with method = “euclidean.” Data at each time point were derived from a single sample.
5
Sp1-Knockdown Impacts A549 Cell miRNA
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Total RNA was extracted using TRIzol from scramble- or Sp1-knockdown A549 cells. The small RNA library preparation and sequencing (single-end 35 bps) using the AB 5500xl SOLiD sequencer were performed by the Center for Bioinformatics and Digital Health at the National Cheng Kung University. The raw reads were preprocessed by performing adapter trimming and then aligned to miRNA retrieved from miRbase (https://www.mirbase.org/ ) using the CLC Genomics Workbench version 10.1.1 software (Qiagen). The OncoMir database (http://www.oncomir.org/ ) was used to identify the targeting genes of miRNAs.
6
Genomic Profiling of Microbial Isolates
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Genomic DNA was isolated using the MoBio Ultraclean Microbial Genomic DNA Isolation Kit (Qiagen, Valencia, CA). DNA libraries were prepared for WGS using Bioo Scientific NEXTflex Rapid DNA-Sequencing kit (Austin, TX) according to the manufacturer’s guidelines. WGS was performed using a MiSeq (Illumina, San Diego, CA) platform instrument using 150-bp paired-end sequencing. De novo assembly of the isolates and multi-locus sequence typing (MLST) was performed using the CLC Genomics Workbench, version 10.1.1 (Qiagen). MLST sequence types were compared using eBurst analysis (www.phyloviz.net ). Accessory gene regulator (agr) groups I-IV were assigned for each isolate using agrD [22 (link)]. Assembled reads derived from raw WGS data for each isolate were interrogated for the presence of MSCRAMM (clfA, clfB, cna, ebh, efb, fnbpA, fnbpB, isdA, isdB, sdrC, sdrD, sdrE) (Table 1 ), biofilm-associated (icaA, icaB, icaC, icaD), and Panton-Valentine leukocidin (PVL) (lukSF-PV) genes using the Microbial Genomics module (version 2.5.1) of CLC Genomics Workbench. Criteria for gene presence was defined by 90% gene identity and a minimum 80% gene length. For surface protein genes with repetitive sequences (clfA, clfB, cna, ebh, sdrC and sdrD), the minimum gene length threshold was adjusted to 60%.
7
Characterization of Bacteriophage ZCEC5
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Bacteriophage ZCEC5 was examined using transmission electron microscopy at the National Research Center (Cairo, Egypt) as previously described (Atterbury et al. 2003 (link)). Briefly, fixed phages on Pioloform grids using glutaraldehyde were negatively stained with 0.5% uranyl acetate. After drying, the specimens were examined using a JEOL 100CX transmission electron microscope.
Genomic DNA was extracted from a lysate of phage ZCEC5 (1010 PFU mL−1) treated with proteinase K (100 μg mL−1 in 10 mM EDTA at pH 8) before purification by the Wizard DNA kit (Promega, UK) according to the manufacturer’s instructions. The genome DNA of phage ZCEC5 was sequenced from libraries prepared using the Illumina tagmentation protocol on the MiSeq platform. The data was composed of 0.52 million paired-end sequence reads with read lengths of approximately 250 bp. The data was de novo assembled using CLC Genomics Workbench version 10.0.1 (Qiagen, Aarhus, Denmark). The open reading frames (ORFs) were predicted from PHASTER (Arndt et al. 2016 (link)). The genome DNA sequence appears in GenBank under the Accession Number MK542015.
Genomic DNA was extracted from a lysate of phage ZCEC5 (1010 PFU mL−1) treated with proteinase K (100 μg mL−1 in 10 mM EDTA at pH 8) before purification by the Wizard DNA kit (Promega, UK) according to the manufacturer’s instructions. The genome DNA of phage ZCEC5 was sequenced from libraries prepared using the Illumina tagmentation protocol on the MiSeq platform. The data was composed of 0.52 million paired-end sequence reads with read lengths of approximately 250 bp. The data was de novo assembled using CLC Genomics Workbench version 10.0.1 (Qiagen, Aarhus, Denmark). The open reading frames (ORFs) were predicted from PHASTER (Arndt et al. 2016 (link)). The genome DNA sequence appears in GenBank under the Accession Number MK542015.
8
Whole-genome sequencing of M. tuberculosis
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Genomic DNA from M. tuberculosis isolates was extracted as per standard protocol [11 (link)]. Libraries were constructed using the Nextera XT DNA preparation kit (Illumina, San Diego, California) and genome sequencing was performed on NextSeq500 (Illumina) with 2 × 150-bp paired-end chemistry. Sequencing reads were mapped to the reference genome M. tuberculosis H37Rv (GenBank NC_000962) and single nucleotide polymorphism (SNP) variants identified using CLC Genomics Workbench version 10.0.1 (Qiagen, Denmark). Reads were processed through the RedDog pipeline (https://github.com/katholt/RedDog ) and Snippy v3.1 and screened for mutations associated with drug resistance.
9
Genomic Assembly and Annotation
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Sequence reads were trimmed and filtered according to quality criteria, and assembled de novo using CLC genomics workbench version 10.0.1 (QIAGEN Aarhus, Denmark). The generated contigs were submitted for annotation in Prokka v.1.12 [70 (link)].
10
Differential Gene Expression Analysis
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Differences were assessed using Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction. The chi-square test was used to evaluate associations between categorical variables. When the p-value was p < 0.05 with chi-square test, residual analysis was performed to identify which category was significant. Gene expression was normalized using transcript per million. Wald test with Benjamini-Hochberg multiple test correction was used for evaluation of differential gene expression. Genes with false discovery rate adjusted p-values < 0.05 were considered differentially expressed. Adjusted residuals were calculated with js-STAR ver 9.1.7 [26 ], evaluation of differential gene expression was performed using CLC Genomics Workbench version 10 (Qiagen), and the other statistical analyses were performed using STAT view ver5.0 (SAS Institute Inc. Cary, NC, USA).
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