Transcripts were profiled for Brucella-infected and bacterial DNA-transfected BMDMs from STING KO and C57BL/6 mice. Total RNA was isolated from BMDMs with the RNeasy RNA extraction kit (QIAGEN) and analyzed by Bioanalyzer RNA 6000 Nano (Agilent Technologies). Gene array analysis was examined by Illumina Sentrix BeadChip Array (Mouse WG6 version 2 for RNA extracted from BMDM) (Affymetrix) at the Oncogenomics Core Facility, University of Miami. Microarray data based on Affymetrix Mouse 2.0 ST platform were normalized using the Robust MultiChip Averaging (RMA) algorithm as implemented in the Bioconductor package Affy. The probes were annotated using Bioconductor annotation package mogene20sttranscriptcluster.db. Fold change was used to compare each pair of microarray samples. The heatmap was generated by R package ggplot2. Microarray analysis was performed at the Center of Computational Science, University of Miami, Florida, USA. GEO accession number is GSE96071(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=uvcjkweipvyzhyb&acc=GSE96071 ).
Bioanalyzer rna 6000 nano
Manufactured by Agilent Technologies
Sourced in United States, Australia
The Bioanalyzer RNA 6000 Nano is a lab equipment product from Agilent Technologies. It is a microfluidics-based platform that provides automated analysis of RNA samples. The device measures the size and concentration of RNA molecules in a sample using electrophoresis and fluorescence detection.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Rneasy rna extraction kit, by Qiagen (1 mentions)
Rneasy mini spin column, by Qiagen (1 mentions)
Genespring gx 14, by Agilent Technologies (1 mentions)
Low input quick amp labeling kit, by Agilent Technologies (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Agilent c scanner, by Agilent Technologies (1 mentions)
Feature extraction 10, by Agilent Technologies (1 mentions)
Pico kit, by Agilent Technologies (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Novaseq600, by Illumina (1 mentions)
15 protocols using bioanalyzer rna 6000 nano
1
Transcriptomic Profiling of Brucella-infected and DNA-transfected BMDMs
2
RNA Extraction and qPCR Analysis
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RNA was extracted by homogenization of liver tissue together with Trizol reagent (Invitrogen). The RNA quantity was evaluated using the NanoDrop ND-1000 UV–Vis Spectrophotometer (Saveen & Werner, Limhamn, Sweden), and RNA quality was tested on a random selection of samples using a BioAnalyzer—RNA 6000 Nano (Agilent Technologies, Santa Clara, CA, USA). Reverse transcription and real-time qPCR analysis were performed as described [36 (link)]. Primer sequences are available on request.
3
Transcriptome Analysis of Perivascular Stem Cells
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RNA was extracted from the cultured G-SCs and P-SCs by miRNeasy micro kit (Qiagen, Inc.) and quantified using NanoDrop One (NanoDrop; Thermo Fisher Scientific, Inc) and BioAnalyzerRNA6000 Nano (Agilent Technologies, In). cDNA synthesis, cRNA labeling and amplification were conducted using the Low Input Quick Amp Labeling Kit (Agilent Technologies, Inc.), and the purification of labeled cRNAs was conducted using RNeasy mini spin column (Qiagen, Inc.). Finally, the microarray (SurePrint G3 Human 8×60k ver.3.0; Agilent Technologies, Inc.) was scanned using a G4900DA Microarray Scanner (Agilent Technologies, Inc.). The differentially expressed genes (DEGs) in P-SCs compared with those in G-SCs were analyzed using GeneSpring GX14.9.1 (Agilent) with a cut-off value of LogFC >1 (ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE174595). Gene Ontology (GO) enrichment analysis was performed to define the biological process of upregulated (up)-DEGs in P-SCs using Cytoscape 3.7.2 (cytoscape.org/) with a cut-off value of adjusted P<0.05. The protein-protein interaction (PPI) network was produced to identify the hub genes using STRING (string-db.org/) and cytoHubba (version 0.1, cytoscape.org/apps/cytohubba) plugin for Cytoscape 3.7.2 with the cut-off value of combined score >0.4.
4
Gene Expression Microarray Analysis Protocol
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Gene expression microarray experiments were performed by DNA Chip Research Inc (Tokyo, Japan). After obtaining the genomic DNA-free total RNA described above, the RNA quantity and quality were verified with a NanoDrop 1000 (Thermo Fisher Scientific), Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and Bioanalyzer RNA6000 Nano (Agilent, Santa Clara, CA). After complementary RNA (cRNA) was synthesized, the cRNA was hybridized with SurePrint G3 Human GE microarray 8 × 60 K (Design ID: 39,494). To scan the microarray image, an Agilent C scanner (Agilent) was used. To quantify the fluorescence intensity, Feature Extraction 10.7.3 software (Agilent) was used. All the data were uploaded to GEO (Gene Expression Omnibus) as GSE120772.
The statistical test was performed using Welch’s t-test with unpaired, unequal variance. Each p-value was calculated asymptotically. To calculate the FDR, the Benjamini–Hochberg correction was applied. Clustering analysis was performed on all the microarray probes using “pvclust” function with method.hclust = “average,” method.dist = “correlation,” nboot = 100 in R program.
The statistical test was performed using Welch’s t-test with unpaired, unequal variance. Each p-value was calculated asymptotically. To calculate the FDR, the Benjamini–Hochberg correction was applied. Clustering analysis was performed on all the microarray probes using “pvclust” function with method.hclust = “average,” method.dist = “correlation,” nboot = 100 in R program.
5
RNA-seq Methodology for Differential Expression
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RNA was isolated by the Qiagen RNeasy Micro Kit including on-column DNA digestion using the RNase-Free DNase Set (Qiagen), according to the manufacturer’s instructions. Quality of total RNA was determined using the Bioanalyzer RNA 6000 Nano or Pico Kit (Agilent Technologies). Bulk RNA sequencing was performed as described before (30 (link)). Genes with a false discovery rate (FDR)-corrected P value < 0.05 were considered as significantly differentially expressed. The GSEA was done using the R-package fgsea (52 (link)). The threshold for significantly enriched gene sets was set to a (FDR) corrected P value < 0.05. The permutation analysis was performed based on custom R-code according to Ostkamp et al. (53 (link)). Distribution-free permutation tests (1 × 107 random permutations) were employed to corroborate metabolic and Th17 pathogenicity gene enrichment in investigated groups. Gene lists associated with Th17 pathogenicity and metabolism were compiled based on literature research and summarized in Dataset S17 . Gene rankings were based on custom R-code and the R-package dplyr (54 ). Lists of all Differentially expressed genes (DEGs) per comparison are provided in Datasets S18–S22 .
6
RNA Isolation and Transcriptome Analysis
7
Tissue Collection for RNA Extraction
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Seven-week-old naïve mice were killed by cervical dislocation between 8 a.m. and 11 a.m. FCx and Hpc were dissected on a chilled Petri dish. FCx included 2 mm of the anterior part of the cortex and the Hpc was dissected whole. Samples were frozen in liquid nitrogen and stored at −80°C. RNA was extracted with TriReagent (Invitrogen) according to the manufacturer’s instructions, followed by quality control (Bioanalyzer RNA 6000 Nano, Agilent).
8
Mouse RNA-seq Library Preparation
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RNA integrity was determined using Bioanalyzer RNA 6000 Nano (Agilent Technologies), according to the manufacturer’s instruction. The RNA samples had RIN values between 8.1 and 8.7. Samples were sequenced at the Norwegian Sequencing Centre (Oslo University Hospital, Ullevål, Oslo). Total RNA samples were subjected to Strand-Specific TruSeqTM mRNA-seq library preparation, and 50 bp paired-end reads were sequenced on an Illumina Novaseq 600 instrument.) To remove/trim low-quality reads and adapter sequences we used BBDuk (BBMap v34.56 [22 ]. Reads were mapped to the Mus musculus reference genome (ENSEMBL release 101, GRCh38.101) using HiSat2 v.1.2.1 [23 (link)]. Read counting was done with FeatureCounts v1.4.6-p1 [24 (link)]. The average read count was 40 million/sample. Raw sequencing data and normalized counts are available in the GEO repository (GSE188546).
9
Efficient RNA Extraction from Frozen Tissue
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Frozen tissue samples were pulverized in Covaris TT1XT using Covaris cryoPREP Impactor and transferred to Lyse Matrix D tubes containing 1.4 mm (about 0.06 in) ceramic spheres (MP Biomedicals). The samples were homogenized after the addition of RLT plus buffer (RNeasy Plus Mini Kit, Qiagen) with beta‐mercaptoethanol (1:100 dilution; Sigma) for 2 × 20 sec at 6800 rpm with a 30 sec break between cycles. RNA was isolated from the resulting homogenate using the RNeasy Plus Mini Kit (Qiagen). RNA concentrations were measured on a Nanodrop instrument (ThermoFisher). RNA samples were desalted and concentrated by precipitation with a 1/10 volume of 5 M NaCl and 7/10 volume of 2‐propanol (Fluka) followed by centrifugation at 20000 × g for 30 min at 4°C. RNA‐containing pellets were washed once with 70% EtOH (Sigma), centrifuged at 20000 × g for an additional 15 min at 4°C, air‐dried, and resuspended in double‐distilled water. RNA quality was determined using an Agilent Bioanalyzer RNA 6000 Nano (minimum RIN value of 7).
10
RNA Extraction and RNAseq Analysis
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An RNA extraction kit (Macherey-Negel) was used to extract total RNA from 10 mg of lyophilized tissue, followed by quality assessment by a Bioanalyzer RNA 6000 nano (Agilent). RNAseq analysis was performed by Lexogen GmbH using QuantSeq 3′-mRNA library preparation and QuantSeq 3′-UTR NextSeq SR75 sequencing. To perform RNAseq analysis, three biological replicates for two time points of 30 min and 6 h were used in each experiment. MapMan software (Usadel et al., 2009) was used to visualize perturbations in gene expression.
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