Total RNA was extracted from cells as described [18 (link)]. RNA concentration was determined with a NanoDropOne spectrophotometer and quality assessed with an Agilent TapeStation4200 (Agilent Technologies, Santa Clara, CA, USA). High-quality RNA from three independent purifications for each experimental point was used for library preparation. Indexed libraries were prepared from 1 μg/ea. of purified RNA with TruSeq Stranded mRNA Library Prep (Illumina, San Diego, CA, USA) following suppliers. Libraries were quantified using the TapeStation 4200 (Agilent Technologies) and Qubit fluorometer (Invitrogen Co., Waltham, MA, USA); then, they were pooled such that each index-tagged sample was present in equimolar amounts. The pooled samples were subjected to cluster generation and sequencing using an Illumina NovaSeq6000 (Illumina) in a 2 × 75 paired-end format. RNA sequencing data have been deposited in the EBI ArrayExpress database (http://www.ebi.ac.uk/arrayexpress , accessed on February 2023) with Accession Number GSE223853.
Nanodrop one spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The NanoDrop One Spectrophotometer is an analytical instrument designed for the measurement of nucleic acid and protein concentrations. It utilizes a unique sample retention system that requires only a small sample volume to perform accurate spectrophotometric analyses.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Novaseq 6000, by Illumina (3 mentions)
Qubit 3.0 fluorometer, by Agilent Technologies (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna library prep, by Illumina (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Purelink, by Thermo Fisher Scientific (1 mentions)
Agilent tapestation, by Agilent Technologies (1 mentions)
T4 rna ligase 1 buffer, by New England Biolabs (1 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Smart seq stranded kit, by Takara Bio (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
10 protocols using nanodrop one spectrophotometer
1
Transcriptome Profiling of Cellular RNA
2
Flubendazole Treatment Transcriptomics in Liver Cancer Cells
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Huh7, MHCC-97H, and SNU449 cells treated with 0.5 μM flubendazole or DMSO for 72 h were harvested using TRIzol and subjected to mRNA Sequence Analysis (Sinotech Genomics, Shanghai). Total RNA was purified using the Tianmo#TR205-200 kit. The quality of total RNA was determined by Agilent Bioanalyzer 2100 (Agilent technologies, California, USA) and quantified by Qubit®3.0 Fluorometer and NanoDrop One Spectrophotometer. Enrichment of differential gene analysis for KEGG and gene ontology (GO) pathways was performed using GSEA software.
3
Transcriptomic Analysis of Colored Carrot Root Cultivars
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Total RNA was extracted from 14 week old whole roots, of colored carrot cultivars (B493B, R6637, Y9244A, P7262), with three roots (i.e. three biological replicates) per sample set. Total RNA was extracted using the TRIzol Plus RNA Purification Kit (Life Technologies, Carlsbad, CA) following the manufacturer’s protocol. DNA was removed with the ‘DNA free-kit’ provided with the RNA purification kit. RNA quantification was measured on a Nanodrop One Spectrophotometer and quality control was done on an Agilent 2100 Bioanalyzer RNA NanoChip. For each RNA sample, libraries were prepared at the University of Wisconsin-Madison Gene Expression Center and sequenced on an Illumina HiSeq 2000 using 1×100 nt reads. After quality control with FastQC (http://www.bioinformatics.babraham.ac.uk /projects/fastqc/), reads were filtered with Trimmomatic version 0.32 with adapter trimming and using a sliding window of length ≥50 and Phred quality score ≥2831 (link). Reads were mapped against the carrot genome sequence (GenBank accession LNRQ01000000.1) using Bowtie2 (Langmead and Salzberg 2012)37 (link). Illumina reads were mapped against the carrot genome sequence (GenBank accession LNRQ01000000.1) using Rsubread version 1.24.238 (link). Transcript expression was analyzed using the Bioconductor package limma39 (link).
4
Synthesis and Purification of GLuc APIE CVB3 RNA
5
RNA Extraction and Expression Analysis
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Total RNA extraction from Caki-1 and Caki-2 cells was performed using the All-Prep DNA/RNA Mini Kit (Qiagen, 80204) (Qiagen, Germantown, MD, USA) as we have previously described [39 (link),40 (link)]. The RNA concentration and purity were determined through a NanoDrop One Spectrophotometer and 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE, USA). RNA was converted to single-stranded cDNA and purified using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, 11904–018). qPCR reactions using primers targeting CA12 (forward 5′-GGACAAATGGGGACAGGAAGGATCAAG-3′ and reverse 5′-GAGGACATTTCATGCTGACAAAATGAG-3′) and NEFL (forward 5′-ATGAGTTCCTTCAGCTACGA-3′ and reverse 5′-TCAATCTTTCTTCTTAGCTGCTTGTTC-3′), with GAPDH (forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′) as the loading control, were analyzed concurrently on a Bio-Rad CFX96 Real-Time System C1000 instrument (Bio-Rad, Hercules, CA, USA). The qPCR analyses were performed in triplicate and included negative control experiments.
6
RNA-seq Analysis of Human Samples
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Briefly, RNA samples were quantified using a NanoDrop One Spectrophotometer, and the RNA integrity number was determined using an RNA 6000 Nano Kit (Agilent) on an Agilent 2100 Bioanalyzer. Next, RNA sequencing libraries were prepared using the SMART-Seq Stranded Kit (Takara, Shiga, Japan). Since the library was less than 2 nM, we excluded six samples. The libraries were then pooled and sequenced, paired-end 150 bp with NovaSeq6000. Fastq sequence files were aligned with the human reference genome (hg38) using STAR 2.7.8a, and the reads were processed using StrandNGS 4.0 (Agilent). Since the mapping rate was less than 40%, we excluded five samples and analyzed the remaining 69 samples. The read counts for each gene were quantified as TPM.
7
Synthesis and Purification of Circular RNAs
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CircRNAs were synthesized by IVT using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). IVT templates were PCR amplified for 30 cycles using forward and reverse circRNA oligos (Supplementary Table 1 ) and column purified before RNA synthesis. One microgram of circRNA template was used per 20 µl of IVT reaction. Reactions were incubated overnight at 37 °C with shaking at 1,000 r.p.m. with a heated lid. IVT templates were subsequently degraded with 2 µl of DNaseI per IVT reaction for 20 minutes at 37 °C with shaking at 1,000 r.p.m. The remaining RNA was column purified before further enzymatic reactions.
To isolate circRNAs, column-purified RNA was digested with one unit of RNaseR per microgram of RNA for 60 minutes at 37 °C with shaking at 1,000 r.p.m. Samples were then column purified, quantified using a NanoDrop One spectrophotometer and verified for complete digestion using an Agilent TapeStation. In some instances, due to reagent shortages, verification was performed with agarose gel under formamide-based denaturing conditions (NEB, B0363S). In cases of incomplete digestion of linear RNAs, RNaseR digestion was repeated.
To isolate circRNAs, column-purified RNA was digested with one unit of RNaseR per microgram of RNA for 60 minutes at 37 °C with shaking at 1,000 r.p.m. Samples were then column purified, quantified using a NanoDrop One spectrophotometer and verified for complete digestion using an Agilent TapeStation. In some instances, due to reagent shortages, verification was performed with agarose gel under formamide-based denaturing conditions (NEB, B0363S). In cases of incomplete digestion of linear RNAs, RNaseR digestion was repeated.
8
Hepatic Gene Expression Profiling after Metformin Treatment
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To obtain the hepatic gene expression profile after metformin treatment, four mice were randomly selected from each group for high-throughput sequencing. Total RNA from liver tissue was extracted in accordance with the RNeasy Mini kit protocol (Qiagen GmbH). The extracted total RNA was quality inspected using the Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.) and quantified using a Qubit® 3.0 Fluorometer and NanoDrop® One spectrophotometer. TruSeq™ RNA Sample Preparation Kit (Illumina, Inc.) was used to construct a sequencing library. In accordance with the instructions of Illumina NovaSeq 6000 (Illumina, Inc.), reagents for sequencing (NovaSeq 5000/6000, S4 300 Cycle; cat. no. 20012866; Illumina, Inc.) were prepared. Paired-end sequencing was performed (lncRNA ≥200 nt). Clusters were generated by cBot after the library was diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 platform (Illumina, Inc.). The fragments of each gene segment were counted after comparison using the Stringtie software (version: 1.3.0; Johns Hopkins University, Baltimore, MD, USA) and normalized by using TMM (trimmed mean of M values) algorithm (http://www.kegg.jp/ ), before the fragments per kilobase million (FPKM) value of each gene was calculated. High-throughput sequencing results were uploaded onto the GEO database under the accession number GSE137840.
9
Dual RNA Isolation and Depletion
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Total RNA was isolated from either S. aureus -infected macrophages, S. aureus grown in BHI to mid-log-phase (inoculum), or uninfected macrophages using the PureLink RNA minikit (Invitrogen) according to the manufacturer’s instructions. Mechanical sample disruption was performed prior to RNA extraction using lysing matrix B tubes (MP Biomedicals) and a FastPrep-24 instrument at an intensity of 6.5 m/s for 3 × 30 sec with 1 min resting on ice between the runs. Sample lysates were then subjected to PureLink on-column DNase treatment (Invitrogen) to remove DNA contamination according to the manufacturer’s instructions. The quality and quantity of the RNA samples were determined using a NanoDrop One spectrophotometer and an Agilent 2100 Bioanalyzer. DNA-depleted RNA samples were further subjected to rRNA depletion using the Ribo-Off rRNA depletion kit for bacteria and human/mouse/rat (Vazyme). The depletion of both eukaryotic and bacterial rRNA was performed simultaneously, following the manufacturer’s instructions recommended for bacteria and with an additional 1 μL rRNA probe for human/mouse/rat added to the hybridization mix.
10
Leaf RNA Isolation using TRIzol
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Total leaf RNA was isolated from 100 mg of fresh or frozen leaf tissue using TRIzol Reagent (Thermo Fisher Scientific). Briefly, to isolate RNA, leaf tissue was frozen in liquid nitrogen and ground into powder using a mortar and pestle. One mL of TRIzol Reagent (Thermo Fischer Scientific, Waltham, MA) was added to the ground leaf tissue and mixed vigorously by vortexing. The leaf and TRIzol mixture was then shaken at room temperature for 10 minutes, followed by the addition of 200 µL of chloroform. This mixture was then vortexed for 30 seconds and then centrifuged at 12,000g for 15 minutes. The aqueous phase was removed and mixed with one volume of cold isopropanol to precipitate the RNA. RNA pellets were washed using 80% cold ethanol. To isolate RNA from P40 and P100 pellets, 1 mL of TRIzol was added to 100 µL of resuspended pellet, followed by the same procedure as used for leaf RNA isolation. RNA pellets were resuspended in 10 to 12 µL of ultrapure DNase/RNase-free water (Invitrogen) and stored at -80°C. RNA quality and quantity was assessed using either a ThermoFisher NanoDrop One spectrophotometer, or an Agilent 2200 Tape Station.
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