Total RNA was extracted from frozen brain tissue using the RNeasy Plus Mini Kit (Qiagen). RNA quality and quantity were determined with a 2100 Bioanalyzer Instrument (Agilent) using the RNA Nano Chip (Agilent); only samples with a RIN value above 7.0 were included. Libraries were made using the TruSeq RNA Library Prep Kit (Illumina; v2) and sequenced at 10 samples/lane as paired-end 101 base-pair reads on a HiSeq 4000 (Illumina) at Mayo Clinic’s Genome Analysis Core. Subsequently, raw sequencing reads were aligned to the human reference genome (GRCh38) with Spliced Transcripts Alignment to a Reference (STAR; v2.5.2b) [15 (link)]. After alignment, library quality was assessed using RSeQC (v3.0.0) [60 (link)], and gene-level expression was quantified using the Subread package (v1.5.1) [37 (link)]. All analyses described below were performed in R (R Core Team; v3.5.3).
Rna nano chip
Manufactured by Agilent Technologies
Sourced in United States, Germany, Spain, France
The RNA Nano Chip is a lab equipment product designed for the analysis and quantification of RNA samples. It is a microfluidic chip that utilizes electrophoretic separation and fluorescent detection to provide accurate and reproducible results for RNA sample analysis.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (46 mentions)
2100 bioanalyzer, by Agilent Technologies (24 mentions)
Rneasy mini kit, by Qiagen (23 mentions)
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Rnase free dnase set, by Qiagen (7 mentions)
Hiseq 4000, by Illumina (6 mentions)
Rneasy kit, by Qiagen (6 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (6 mentions)
Rneasy plus mini kit, by Qiagen (5 mentions)
Turbo dnase, by Thermo Fisher Scientific (5 mentions)
Nextseq 500, by Illumina (4 mentions)
Rneasy plant mini kit, by Qiagen (4 mentions)
Tri reagent, by Merck Group (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Novaseq 6000, by Illumina (4 mentions)
Truseq stranded mrna library prep kit, by Illumina (4 mentions)
Bioanalyzer, by Agilent Technologies (3 mentions)
123 protocols using rna nano chip
1
RNA Sequencing Workflow for Brain Tissue
2
In Vitro Transcription of eGFP mRNA
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Template for in vitro transcription comprising a T7 promotor followed by a modified 5′-UTR, the coding sequence of enhanced GPF (eGFP from Mycobacterium tuberculosis H37Rv; Gene ID: 20473140), the 3′-UTR of the mouse haemoglobin alpha chain and a restriction site was synthesised by DNA 2.0. Plasmid was linearised by endonuclease restriction and used as template for in vitro transcription using T7 RNA polymerase. The in vitro transcripts were purified using RNeasy Kit (Qiagen), enzymatically capped (Vaccinia capping enzyme), and polyadenylated (poly-A polymerase). After DNase I treatment, mRNAs were again purified using RNeasy Kit (Qiagen). RNA concentrations were determined by measuring the absorbance at 260 nm using a Nanodrop spectrophotometer. The length of the in vitro transcripts and the polyadenylated mRNAs were monitored by Bioanalyzer electrophoreses on a RNA Nano chip (Agilent).
3
High-throughput RNA Sequencing of Gene Expression
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RNA samples were assessed for quality on the Agilent Bioanalyzer with the RNA Nano Chip, providing an RNA Integrity Number. Samples were quantified using the Qubit 2.0 fluorometer and the Qubit RNA Broad Range assay. Ten nanograms of RNA was reverse-transcribed to make cDNA, and then, target genes were amplified for 12 cycles of PCR using the Ion AmpliSeq Human Gene Expression Core Panel. This panel contains 20,802 amplicons (41,604 primers) of approximately 150 bases in length in a single pool. Ion Torrent sequencing adapters and barcodes were ligated to the amplicons, and adapter-ligated libraries were purified using AMPure XP beads. Libraries were quantified by qPCR and diluted to 100 pM. Templates were prepared using the Ion PI Hi-Q OT2 200 Kit and sequenced using the Ion PI Hi-Q Sequencing 200 Kit. The Ion Proton platform was used to analyze the data. Analysis of gene expression was performed using the software package Babelomics 5.0 (53 (link), 54 (link)). Statistical analysis of significance was performed using Benjamini and Hochberg false discovery rate multiple test. GSEA was performed using GSEA 3.0 software from the Broad Institute (www.gsea-msigdb.org ) (55 (link)). Gene ontology analysis was performed using DAVID functional annotation web resource (https://david-d.ncifcrf.gov ).
4
Parathyroid Tumor RNA Profiling
5
Isolation and Quality Assessment of Total RNA
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Total RNAs were isolated from the bone marrow/peripheral blood/cells according to manufacturer’s recommendation (Qiagen RNeasy mini kit). DNA digestion was performed to ensure that the RNA was free of DNA contamination (Qiagen DNase I, Hilden, Germany). The integrity of the total RNAs used for gene expression microarray were checked with Bioanalyser (RNA Nano Chip, Agilent 2100 Bioanalyser). All samples included in the gene expression microarray had an RNA integrity number (RIN) of at least 8.0. The purity of the isolated Total RNAs was also checked with NanoDrop ND-1000 UV–VIS spectrophotometer to ensure that the purity was within the range of 1.80–2.10.
6
Bovine Transcriptome Profiling via RNA-Seq
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Total RNA was extracted using a Tempus™ Spin RNA Isolation Kit (Thermo-Fisher Scientific), per the manufacturer’s directions. Following isolation, all further RNA work was completed using the facilities at the OSU Center for Genome Research and Biocomputing (Corvallis, OR, USA). An Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and an RNA Nano Chip (Agilent Technologies) were used to quantify RNA quality and concentration. All samples for cDNA library prep had satisfactory RNA Integrity Numbers of ≥ 8.0.
Strand-specific cDNA libraries were prepared with poly-A selection for each sample, checked with an Agilent TapeStation 4200 (Agilent Technologies), and validated with single dilution qPCR. Each library was sequenced using an Illumina HiSeq 3000 (Illumina, Inc., San Diego, CA, USA) with a 100 bp read length (single end). Pseudoalignment of each cDNA library to the Bos taurus reference genome (UMD3.1.1) [34 (link)] was completed using kallisto v. 0.42.4 [35 (link)] and converted to gene-level estimates through the tximport R package [36 (link)].
Strand-specific cDNA libraries were prepared with poly-A selection for each sample, checked with an Agilent TapeStation 4200 (Agilent Technologies), and validated with single dilution qPCR. Each library was sequenced using an Illumina HiSeq 3000 (Illumina, Inc., San Diego, CA, USA) with a 100 bp read length (single end). Pseudoalignment of each cDNA library to the Bos taurus reference genome (UMD3.1.1) [34 (link)] was completed using kallisto v. 0.42.4 [35 (link)] and converted to gene-level estimates through the tximport R package [36 (link)].
7
Microdissecting mPFC Tissue for RNA Isolation
8
Transcriptome analysis of P. patens SOG1 mutants
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For transcriptomic analysis, the protonema tissues of WT, sog1a‐2, sog1b‐1, and sog1a‐2 sog1b‐2 were suspended in 10 ml of water and homogenized. About 0.25 OD730 of the homogenized tissues was spread on BCDAT agar medium overlaid with cellophane and cultured for 13–14 days under 16‐h white light–8‐h dark cycle at 23°C. The tissues were exposed to 60Co γ‐rays for 15 min with a dose rate of 800 Gy h−1 at TARRI. After the irradiation, the tissues were incubated under white light at 23°C for 45 min and then immediately frozen with liquid nitrogen.
About 100 mg of protonema tissues were ground with liquid nitrogen, and total RNAs were extracted using RNeasy PowerPlant Kit (QIAGEN). The RNAs were treated with RNase‐Free DNase Set (QIAGEN), followed by column purification to remove genomic DNA contamination. RNA quality was checked using an RNA 6000 Bioanalyzer Kit and RNA Nano Chip (Agilent Technologies). For library preparation, mRNA was extracted from the total RNA, and libraries were prepared using a TruSeq Stranded mRNA LT Sample Kit (Illumina). The pooled libraries were sequenced on a NextSeq 500 (Illumina) to obtain the single‐end reads of 76 bp. The obtained reads were mapped to the P. patens reference genome (v.3.3). The count data were subjected to a trimmed mean of M‐value normalization in EdgeR (McCarthy et al., 2012 (link); Robinson et al., 2010 (link)).
About 100 mg of protonema tissues were ground with liquid nitrogen, and total RNAs were extracted using RNeasy PowerPlant Kit (QIAGEN). The RNAs were treated with RNase‐Free DNase Set (QIAGEN), followed by column purification to remove genomic DNA contamination. RNA quality was checked using an RNA 6000 Bioanalyzer Kit and RNA Nano Chip (Agilent Technologies). For library preparation, mRNA was extracted from the total RNA, and libraries were prepared using a TruSeq Stranded mRNA LT Sample Kit (Illumina). The pooled libraries were sequenced on a NextSeq 500 (Illumina) to obtain the single‐end reads of 76 bp. The obtained reads were mapped to the P. patens reference genome (v.3.3). The count data were subjected to a trimmed mean of M‐value normalization in EdgeR (McCarthy et al., 2012 (link); Robinson et al., 2010 (link)).
9
RNA Quality and Quantity Assessment
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The total RNA samples were checked for quality and quantity on an Agilent 2100 Bioanalyzer using an RNA Nano chip (part # 5067-1511).
10
Transcriptome Analysis of HepG2-NTCP Cells
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HepG2-NTCP cells were cultured with D10/2%DMSO or HMM for 24 h, followed by total RNA extraction with RNeasy Isolation kit (Qiagen) and treated with DNase I. Following assessment of the quality of total RNA using an Agilent 2100 Bioanalyzer and RNA Nano chip (Agilent Technologies), 150 ng total RNA was treated to deplete the levels of ribosomal RNA (rRNA) using target-specific oligos combined with rRNA removal beads. Following rRNA removal, mRNA was fragmented and converted into double stranded cDNA. Adaptor-ligated cDNA was amplified by limit cycle PCR. After library quality was determined via Agilent 4200 TapeStation and quantified by KAPA qPCR, approximately 60 million paired-end 150 bp sequence reads were generated on the Illumina HiSeq 4000 platform. For data analysis, each sample was aligned to the GRCh38.p13 assembly of the human reference from GENCODE using version 2.1.0 of the RNA-Seq aligner HISAT2. Features were identified from the GTF file from GENCODE. Feature coverage counts were calculated using fetureCounts and differentially expressed features were calculated using DESeq2 package in R and significantly differential expressions were those with a false discovery rate (FDR) of < 0.05 and fold-change of > 2.
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