Total RNA was isolated using a RNAprep Pure Plant Kit and treated with DNase I (Tiangen, Beijing, China) according to manufacturer’s instructions. RNA concentrations and quality were determined by a Thermo 2000 Bioanalyzer with a RNA NanoDrop (Thermo Scientific, USA). Library preparation for RNA-Seq was conducted using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Cat. RS-122-2101, USA) according to manufacturer’s protocol. And the final libraries were quantified by QuantiFluor dsDNA System (Promega, USA) and sequenced on an Illumina HiSeq 2500 platform (Illumina Inc. USA) at Berry Genomics Corporation, Beijing, China.
Thermo 2000 bioanalyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo 2000 Bioanalyzer is a laboratory instrument designed for the analysis of biomolecules. It utilizes microfluidic technology to perform automated electrophoretic separation and detection of DNA, RNA, and proteins. The core function of the Thermo 2000 Bioanalyzer is to provide rapid and precise quantification and qualification of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Rna nanodrop, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Tiangen Biotech (2 mentions)
Truseq stranded mrna lt sample prep kit, by Illumina (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Quantifluor dsdna system, by Promega (2 mentions)
Rna nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Super smart pcr cdna synthesis kit, by Takara Bio (1 mentions)
Rnaprep pure plant plus kit, by Tiangen Biotech (1 mentions)
Iq5 real time pcr system, by Bio-Rad (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
4 protocols using thermo 2000 bioanalyzer
1
RNA Extraction and RNA-Seq Library Preparation
2
Quantifying Gene Expression in Developing Tea Leaves
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Total RNA from developing tea leaves or treatment samples was extracted by using RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. RNAs quality was checked using a Thermo 2000 Bioanalyzer and an RNA NanoDrop ND-2000 Spectrophotometer (Thermo Fisher Scientific, Shanghai, China), and the first-strand cDNA synthesis was done with the Super SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA). Quantitative real-time PCR (qRT-PCR) was conducted using the SYBR Green PCR products (Yushen, Shanghai, China). Gene-specific primers provided in Table S9 (see online supplementary material) were used for qRT–PCR in 96-well plates (iQ5 Real Time PCR System; Bio-Rad), as described previously. The ETF and β-ACTIN genes were used as the internal reference to calculate relative gene expression [27 (link), 55 (link)]. All analyses were performed in three biological replicates with three technical replications.
3
Transcriptional Analysis of Gsb-Resistance Genes
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Leaves from HS and XH plants after inoculation (0, 0.5, 1, 2, 3, 5, and 7 days) were sampled and frozen in liquid nitrogen. Total RNA was isolated from leaves using mirVanaTM miRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. RNA concentration and quality were assessed using a Thermo 2000 Bioanalyzer with an RNA NanoDrop (Thermo Scientific, USA; http://www.thermo.com (23 August 2017, date last accessed)). Samples showing A260/A230 ratio of 2.0–2.2 and A260/A280 ratio of 1.8–2.0 were used for further analysis. For quantitative real-time PCR, 1 µg of total RNA was reverse transcribed to first-strand cDNA in a final reaction volume of 20 µl using miScript II RT Kit (Qiagen). The transcriptional patterns of the candidate Gsb-resistance genes were analysed using a quantitative real-time PCR (RT-qPCR). Details regarding the primers used in this study are provided in Supplementary Table S10 .
4
RNA-Seq Library Preparation Protocol
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Total RNA was isolated using a RNAprep Pure Plant Kit and treated with DNase I (Tiangen, Beijing, China) according to manufacturer's instructions. RNA concentrations and quality were determined by a Thermo 2000 Bioanalyzer with a RNA NanoDrop (Thermo Scientific, USA). Library preparation for RNA-Seq was conducted using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Cat. RS-122-2101, USA) according to manufacturer's protocol. And the final libraries were quantified by QuantiFluor dsDNA System (Promega, USA) and sequenced on an Illumina HiSeq 2500 platform (Illumina Inc. USA) at Berry Genomics Corporation, Beijing, China.
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