Following tissue collection as described above, total RNA was isolated using Ambion RNAqueous total RNA Isolation Kit and assayed using Agilent RNA 6000 Nano Bioanalyzer kit/instrument. Stranded mRNA libraries were prepared using TruSeq Stranded mRNA kits. Eight to 12 samples per lane were pooled and sequenced on an Illumina HiSeq 4000 instrument using a single-end 50 bp protocol. Reads were aligned to mouse genome (mm9) using STAR (version 2.4.2a) (Dobin et al., 2013 (link)) and gene counts produced using featureCounts (Liao et al., 2014 (link)) and mm9 knownGenes. Quality assayed using FastQC (Andrews, 2010 ) and RSeQC (Wang et al., 2012 (link)), and samples that exhibited 3′ bias or poor exon distribution were discarded. Raw RNA-seq fastq files and a gene count matrix is available on GEO GSE166376 .
Rna 6000 nano bioanalyzer
Manufactured by Agilent Technologies
Sourced in Australia
The RNA 6000 Nano Bioanalyzer is a lab equipment product from Agilent Technologies that is used for the analysis of RNA samples. It provides quantitative and qualitative assessment of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq transcriptome human gene expression kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using rna 6000 nano bioanalyzer
1
RNA-Seq of Mouse Tissue Samples
2
Quantitative RT-PCR Analysis of PI3K/AKT/mTOR Pathway
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Total RNA was extracted from 1x107-treated UVECS using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The purity of the RNA was determined using an RNA 6000 nano-bioanalyzer (Agilent Technologies, Inc.). Total RNA was reverse transcribed into cDNA using the SuperScript™ II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h at 42˚C, according to the manufacturer's protocol. qPCR was subsequently performed using 12.5 µl 2X real-time PCR SYBR®-Green master mix, 1.5 µl cDNA template, 2 µl primers and 9 µl 0.1% DEPC H2O (Fast SYBR-Green Master Mix; Invitrogen; Thermo Fisher Scientific, Inc.). The following primer pairs were used for the qPCR: PI3K forward, 5'-ATTACGCTAGTTACACTGCA-3' and reverse, 5'-TGGACCTGGCCATCGACTGA-3'; AKT forward, 5'-GCCACCATGAATGAGGTGAAT-3' and reverse, 5'-GCGTATGACAAAGGTGTTGGG-3'; mTOR forward, 5'-ACTCGCTTCTATGACCAACTGA-3' and reverse, 5'-TTTCCATGACAACTGGGTCATTG-3'; and β-actin forward, 5'-CAACGAGCGGTTCAGGTGT-3' and reverse, 5'-TGGAGTTGAAGGTGGTCTCGT-3'. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 30 sec; 45 cycles of 95˚C for 30 sec, 56.8˚C for 30 sec and 72˚C for 30 sec. Expression levels were normalized to the internal reference gene β-actin. Expression levels were quantified using the 2-ΔΔCq method (27 (link)).
3
Quantitative Real-Time PCR of RNA Splice Variants
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Total RNA was prepared from freshly purified cell populations or cells growing in exponential growth phase using TRIzol reagent as per the manufacturer's instructions (Thermo). Integrity and quantity of extracted RNA were assessed using an RNA 6000 Nano Bioanalyzer (Agilent Technologies, Mulgrave, Victoria, Australia). All RNA used had a RNA integrity number > 8.8. For cDNA, 100 ng of DNase I (Thermo)‐treated RNA was reverse‐transcribed into cDNA using SuperScript III (Thermo). Oligonucleotide primers designed to detect splice variants were checked for specificity by BLAST alignment and are listed in the supplementary material. Gene expression was performed by qPCR on the cDNA using optimized primers and Fast SYBR® Green Master Mix (Thermo). Duplicate samples of cDNA were amplified using a 7500 Fast Real‐time PCR System (ABI). CT values for splice variant amplification were normalized to the HPRT endogenous gene and presented as fold changes to a CD14+ or U937 cDNA reference sample using the formula: fold change = 2−ΔΔCT (Pfaffl, 2001 ). Primer efficiencies were all greater than 98%.
4
RNA Integrity and Transcriptome Analysis
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RNA integrity was measured on the RNA 6000 Nano Bioanalyzer (Agilent Technologies, Inc.) and the RNA integrity number (RIN) was used as a parameter to define sample quality. RNA samples with an RIN equal to or greater than 7 were also quantified by Qubit Fluorometer (RNA HS Assay Kit - Invitrogen) and reverse transcribed using a Vilo SuperScript cDNA Synthesis Kit, according to the manufacturer’s instructions. The Reverse Transcription Master Mix used included: 5× Vilo Reaction Mix and 10× SuperScript Enzyme Mix. Briefly, 10 ng total RNA was reverse transcribed by adding 4.5 μL Master Mix to the samples. Barcoded libraries were generated using an Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific, Waltham, MA). The resulting libraries were then used for downstream template preparation on the Ion Chef System and sequencing occurred on the Ion S5 System, according to the manufacturer’s instructions.
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