Total RNA was isolated using Trizol reagent (Ambion) and genomic DNA was removed with the TURBO DNase I kit (Ambion). RNA concentration and quality was assessed with the Agilent Bioanalzyer (Agilent, Santa Clara, CA). Library construction was prepared using the TruSeq RNA sample Preparation Kit (Illumina) and sequenced using an Illumina HiSeq2500 (Illumina, Santa Clara, CA) with a read depth of 20-25 million reads at 1×100bp. Details on analysis of RNA-sequencing data are described in the Supplementary Methods .
Agilent bioanalzyer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Bioanalyzer is a microfluidics-based platform used for the analysis of biomolecules, such as DNA, RNA, and proteins. It provides automated, rapid, and sensitive quantification and quality assessment of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Turbo dnase 1 kit, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Bcl2fastq2, by Illumina (1 mentions)
Kapa library quantification kit, by Illumina (1 mentions)
Kapa library preparation kit, by Roche (1 mentions)
Nextseq 500 v2 sequencer, by Illumina (1 mentions)
3 protocols using agilent bioanalzyer
1
RNA Isolation and RNA-seq Sample Preparation
2
Whole-Exome Sequencing Library Preparation
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Sequencing libraries were generated from 1 μg of DNA using the Kapa Library Preparation Kit (Kapa Biosystems, Inc., Wilmington, MA, USA). Library DNA size and quality parameters were evaluated with the Agilent BioAnalzyer (Agilent Technologies, Santa Clara, CA, USA), and equimolar amounts were used for whole-exome enrichment using the Roche NimbleGen SeqCap EZ Exome Library v3.0 kit, which targets 64 Mb of genomic DNA, covering over 20,000 genes (Roche NimbleGen, Inc., Madison, WI, USA). Each library was quantitated with the Kapa Library Quantification Kit and each enriched DNA library was sequenced on an Illumina NextSeq 500 v2 sequencer to generate approximately 100 million 75-base paired-end reads for a final average target coverage depth of 100X. The raw sequence data were demultiplexed using the Illumina bcl2fastq2 software (Illumina, Inc., San Diego, CA, USA).
3
Bulk RNA-seq Differential Expression Analysis
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Total RNA was isolated using TRIzol reagent (15596026; Thermo Fisher Scientific, Waltham, MA, USA) and genomic DNA was removed with the TURBO DNase I kit (AM2238; Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration and quality were assessed with the Agilent Bioanalzyer (Agilent, Santa Clara, CA). Library construction was prepared using the TruSeq RNA sample Preparation Kit (Illumina), following the manufacturer's protocol for RNA input quantity relative to RNA quality. Sequencing was performed on Illumina HiSeq2500 (Illumina, Santa Clara, CA) to generate 100-bp paired-end reads. DESeq2 R package was used to normalize the original read count matrix for a unified expression level and define the differential expression of genes (DEGs). If the criterion of |log2 (fold change)| > 1 and P < 0.05, we could consider it statistically significant. Principal Component Analysis (PCA) was then performed. We converted Ensembl ID (downloaded from http://asia.ensembl.org ) into gene names with Perl language. Heatmaps were created by the pheatmap R package. R software (version 4.0.5) and the limma R package were employed to deal with data. Images were generated by the ggplot2 R package. GO and KEGG enrichment was implemented on these upregulated DEGs with clusterProfiler R package.
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