RNA was extracted from 500-mg rind sample aliquots without prior separation of microbial cells, as previously described (Monnet et al., 2012 (link)), except that the DNase treatment was performed on the RNeasy spin columns (Qiagen, Courtaboeuf, France). Purified RNA was quantified with Qubit RNA assay kits on the Qubit 3.0 fluorimeter (ThermoFischer Scientific). RNA quality was analyzed with an Agilent model 2100 Bioanalyzer (Palo Alto, CA, USA) using RNA 6000 NANO chips, according to the manufacturer's instructions. For each sample, 10 μg of total RNA was then subjected to rRNA depletion using the Epicentre Ribo-Zero™ Magnetic Gold Kit (Tebu-bio, Le Perray-en-Yvelines, France) for bacteria (reference MRZB12424) and for yeasts (reference MRZY1324). Depletion was performed according to the manufacturer's instructions, except that a mixture (50/50) of the yeast and bacteria Ribo-Zero rRNA solutions was used. The rRNA-depleted samples were then purified using a RNeasy MinElute Cleanup Kit (Qiagen), according to the modified procedure described in the Ribo-Zero™ Magnetic Gold Kit technical procedure. The quality of depleted RNA was assessed on an Agilent model 2100 Bioanalyzer, using RNA 6000 PICO chips.
Agilent model 2100 bioanalyzer
Manufactured by Agilent Technologies
Sourced in United States, Canada
The Agilent Model 2100 Bioanalyzer is a lab instrument that provides automated electrophoretic analysis of biomolecules, such as DNA, RNA, and proteins. It uses microfluidic technology to analyze samples with high resolution and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Truseq rna sample prep, by Illumina (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Trizol a reagent, by Tiangen Biotech (3 mentions)
Rnase free dnase 1, by Takara Bio (3 mentions)
Mirvanatm mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Model 450 fluidics station, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000 genome sequencing platform, by Illumina (2 mentions)
Expression console software, by Thermo Fisher Scientific (2 mentions)
Mirvana rna purification kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample prep kit v2, by Illumina (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000 genome sequencer, by Illumina (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Geneamp pcr systems 2700, by Thermo Fisher Scientific (1 mentions)
41 protocols using agilent model 2100 bioanalyzer
1
Microbial RNA Extraction and Depletion
2
RNA Extraction and Reverse Transcription from Whole Blood
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Whole blood was collected from all participants by venipuncture of a forearm vein into PAXgene Blood RNA tubes (Qiagen, California, USA), which were stored at −20°C until processed. Total RNA was extracted using the PAXgene Blood RNA Kit IVD according to the manufacturer’s instructions (Qiagen, California, USA). All samples were eluted in 80 µl of elution buffer and stored at −80°C until further analysis. RNA yield and quality were assessed using an Agilent Model 2100 Bioanalyzer (Agilent Technologies, California, USA) and a DropSense 16 spectrophotometer (TRINEAN, Belgium). All samples had 260/280 > 2.0 and RIN > 7.0.
Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, California, USA), according to the manufacturer’s specifications. Briefly, for each sample, 100 ng of RNA was added to 2 μl of random and oligo (dT) primers in a final reaction of 20 μl. These reaction tubes were then placed in the GeneAmp® PCR Systems 2700 (Applied Biosystems, California, USA) thermocycler at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min to inactivate the reverse transcriptase enzyme. The cDNA samples were then stored at −20°C until analyzed.
Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, California, USA), according to the manufacturer’s specifications. Briefly, for each sample, 100 ng of RNA was added to 2 μl of random and oligo (dT) primers in a final reaction of 20 μl. These reaction tubes were then placed in the GeneAmp® PCR Systems 2700 (Applied Biosystems, California, USA) thermocycler at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min to inactivate the reverse transcriptase enzyme. The cDNA samples were then stored at −20°C until analyzed.
3
RNA-seq of porcine fetal lungs
4
Microarray Analysis of Myeloid Cells
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Purified Gr1− myeloid cells were treated as indicated before (Wu et al, 2017 ) and total RNA was extracted by using the guanidinium isothiocyanate method (TRIzol reagent) followed by purification using the RNeasy Kit (Qiagen). RNA quality was assessed by using the Agilent Model 2100 Bioanalyzer (Agilent Technologies). Ten micrograms of total RNA was processed for analysis on the microarray by using the Affymetrix GeneChip one‐cycle target labeling kit (Affymetrix) according to the manufacturer's protocols. The resultant biotinylated cDNA was fragmented and hybridized to the GeneChip Mouse Gene 2.0 ST Array. The arrays were washed, stained, and scanned using the Affymetrix Model 450 Fluidics Station and Affymetrix Model 3000 scanner according to the manufacturer's protocols. Expression values were generated by using Microarray Suite (MAS) v5.0 software (Affymetrix). Each sample and hybridization underwent a quality control evaluation, including cDNA amplification of > 4‐fold, percentage of probe sets reliably detecting between 40 and 60% present call, and 3′–5′ ratio of GAPDH gene < 3.
5
RNA-Seq Analysis of Primary Airway Cultures
6
Genomic and Transcriptomic Profiling of Tumor Tissues
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Genomic DNA (gDNA) and total cellular RNA were purified from flash frozen tumor (ovarian and endometrial) and normal tissues, as described previously [20 (link)]. Only RNAs with an RNA integrity number (RIN) [21 (link)] greater than or equal to 7.0 were selected for RNA sequencing. Each qualifying tumor was fragmented, converted to cDNA and ligated to bar-coded sequencing adaptors using Illumina TriSeq stranded total RNA library preparation (Illumina, San Diego, CA, USA). The yield and purity of Genomic DNAs were assessed on a NanoDrop Model 2000 spectrophotometer and by using horizontal agarose gel electrophoresis.
Indexed libraries were confirmed on the Agilent Model 2100 bioanalyzer and libraries were then combined into pools for sequencing. Sequencing for both RNA and DNA was then conducted on the Illumina HiSeq 4000 genome sequencing platform using 150 bp paired-end SBS chemistry. All library preparation and sequencing were performed in the Genome Facility of the University of Iowa Institute of Human Genetics (IIHG). Quality control (QC) of RNA sequencing experiments (RNA-seq) and whole genome sequencing (WES) were performed to minimalize technical biases.
Indexed libraries were confirmed on the Agilent Model 2100 bioanalyzer and libraries were then combined into pools for sequencing. Sequencing for both RNA and DNA was then conducted on the Illumina HiSeq 4000 genome sequencing platform using 150 bp paired-end SBS chemistry. All library preparation and sequencing were performed in the Genome Facility of the University of Iowa Institute of Human Genetics (IIHG). Quality control (QC) of RNA sequencing experiments (RNA-seq) and whole genome sequencing (WES) were performed to minimalize technical biases.
7
Isolation and Sequencing of Total RNA from CFBE Cells
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Total RNA was isolated from CFBE cells using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA). Total RNA was tested on an Agilent Model 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA integrity number (RIN) greater than 8.0 were selected for further processing. Libraries were prepared using the TruSeq RNA Sample Prep (Illumina, San Diego, CA, USA) and submitted to the Iowa Institute of Human Genetics Genomics Division for deep sequencing. Data were processed using Kallisto and Sleuth.19 , 20
8
Extraction and Characterization of High-Quality RNA
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JEG-3 and JAR cells were cultured under optimal conditions (JEG-3 in EMEM 10% FBS; JAR in RPMI 1640 10% FBS) and cells for RNA purification were harvested at 80–90% confluence. Placental tissues and endometrial and ovarian tumors were removed from −80°C storage and transitioned in RNAlaterICE (Life Technologies) for 24 to 48 hours prior to RNA extraction. All RNA purifications were performed with the miRvana RNA extraction kit (Life Technologies) according to manufacturers' recommendations. RNA yield and purity were assessed on a NanoDrop Model M-1000 spectrophotometer and an Agilent Model 2100 Bioanalyzer. Only RNAs with an integrity number (RIN) [15 ] of at least 5.0 (range 5.2 to 9.7) were used in this study.
Human fetal tissue total RNAs from brain, heart, liver, and kidney were purchased from Clontech.
Human fetal tissue total RNAs from brain, heart, liver, and kidney were purchased from Clontech.
9
Microarray-based Gene Expression Analysis
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RNA quality was assessed by using the Agilent Model 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). 150nanogram of total RNA was processed for use on the microarray by using the Affymetrix WT plus kit according to the manufacturer’s recommended protocols. The resulting biotinylated cRNA was fragmented and then hybridized to the Clariom D array (Applied Biosystems). The arrays were washed, stained, and scanned using the Affymetrix Model 450 Fluidics Station and Affymetrix Model 3000 7G scanner using the manufacturer’s recommended protocols by the Microarray Facility. Expression values were generated by using Expression Console software (Affymetrix). Each sample and hybridization underwent a quality control evaluation.
10
Transcriptional Profiling of Chromogranin A Knockout in Kidney
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