Two established next-generation sequencing platforms were involved in this study: Illumina RNA-seq and Proton RNA-seq. Both the Proton RNA-seq and AmpliSeq rely on Ion Proton for the sequencing step, though each uses different RNA library preparation protocols (as mentioned above). Illumina RNA-seq and Proton RNA-seq use two next-generation sequencing technologies. Illumina RNA-seq uses the technology of sequencing by synthesis, which requires fluorescence and signal scanning. For this method, prepared libraries are denatured to single strands by linearization. The four nucleotides (GCAT) are coupled to a cleavable fluorescent dye and a removable blocking group, which complements the template one base at a time, yielding a signal to be captured by a charge-coupled device [18 (link)]. Proton RNA-seq is based on semiconductor sequencing technology with emulsion PCR amplified libraries bound to Ion spheres. The template spheres are loaded on a sequencing chip and run on the Proton instrument. The sequencing is then performed by flushing nucleotides individually over the surface of the library-loaded chip. Reads are produced by detecting the change in pH as each nucleotide is incorporated [18 (link), 19 (link)]. In this study, raw reads from Illumina RNA-seq are pair-ended and those from Proton RNA-seq are single-ended.
Rna seq
Manufactured by Illumina
Sourced in United States, China
RNA-Seq is a laboratory equipment designed for the analysis of RNA expression. It utilizes next-generation sequencing technology to capture and quantify the complete transcriptome of a biological sample. The core function of RNA-Seq is to provide a comprehensive profile of gene expression levels across the entire genome.
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Lab products found in correlation
Hiseq 2500, by Illumina (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Hiseq 2000, by Illumina (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Hiseq platform, by Illumina (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Fast super evagreen qpcr master mix, by US Everbright (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq 2000 system, by Illumina (3 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
Novaseq, by Illumina (2 mentions)
Agilent rna 6000 nano chip, by Agilent Technologies (2 mentions)
Bcl2fastq v2, by Illumina (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (2 mentions)
Infinium methylationepic array, by Illumina (2 mentions)
Quick dna rna miniprep kit, by Zymo Research (2 mentions)
Kapa rna hyperprep kit, by Roche (2 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (2 mentions)
148 protocols using rna seq
1
Comparative Analysis of Illumina and Proton RNA-seq
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RNA-Seq Analysis of Murine Liver B Cells
3
Transcriptome Analysis of Potato Stress Responses
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The expressional data for drought (cv. Alegria, Desiree, Milva, Saturna, RNA Seq, Illumina HiSeq2000, GEO: GSM2060109), salinity (150 mM NaCI for 24 h) (cv. DM 1–3 R44, RNA‐Seq, Illumina Genome Analyzer II), mannitol (260 μM for 24 h) (cv. DM 1–3 R44, RNA‐Seq, Illumina Genome Analyzer II), and heat (35°C for 24 h) (cv. DM 1–3 R44, RNA‐Seq, Illumina Genome Analyzer II) treatments were received from Spud DB (http://spuddb.uga.edu/ ) by typing the keyword “GATA” into “Functional Annotation Keyword Search” tool (annotation dataset DM v6.1). The collected data were used to build a heat map using Morpheus software (https://software.broadinstitute.org/morpheus/ ). The rows were hierarchically clustered using default settings.
4
Transcriptomic Analysis of HULK Gene Expression
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Analysis of HULK gene expression pattern by means of RNA-Seq, in situ hybridization and GUS staining are described in Methods S2 (Weigel and Glazebrook, 2002 ; Kover et al., 2009 (link); Gan et al., 2011 (link); Trapnell et al., 2012 (link)).
Transcriptome profiling using Illumina RNA-Seq (http://www.illumina.com/ ) with biological replication for seedlings of Col–0, hua2–7 single mutants, hua2–7 hulk1 double mutants and hua2–7 hulk1 hulk2 triple mutants is described in Methods S3 (Anders and Huber, 2010 (link); Doherty and Kay, 2010 (link); Du et al., 2010 ; Gan et al., 2011 (link); Trapnell et al., 2012 (link)).
Transcriptome profiling using Illumina RNA-Seq (
5
Splice Junction Detection in Transcriptome
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Splice junctions were identified using GMAP for Iso-Seq CFLs and STAR for Illumina reads. Splice junctions detected by both Iso-Seq and Illumina short-read sequencing were considered validated. Because 101-bp reads cannot be assigned definitively to specific splice junctions within the W repeat region, a repeat splice junction was considered to have been detected by Illumina RNA-Seq if any of the set of possible alignments was reported by STAR. Illumina RNA-Seq read depth for repeat splice junctions was normalized by dividing by the number of equivalent genomic alignments possible.
6
Integrating Multi-Omics Data for Alzheimer's Research
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Transcriptomic analysis of Pl and Pc venom glands
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The transcriptomic analysis was performed from samples of 100 Pl and 100 Pc venom glands using Illumina RNA-Seq. To improve de novo assembly for Pl, we also generated Sanger sequences from samples of 50 venom glands, and 454 sequences from full insect bodies of 85 males and 85 females obtained from six siblings (Supplementary Fig. S1 ). Pl and Pc venom glands were dissected in Ringer’s saline and stored at −80 °C. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to manufacturer’s instructions, and quality was checked using an Agilent BioAnalyzer. cDNA library construction for Illumina RNA-Seq and 454 sequencing was performed by Beckman Coulter Genomics (USA). cDNA library used for Sanger sequencing was constructed from 1 μg of total RNA using the Creator SMART cDNA Library Construction Kit (Clontech). Ligation products were transformed into ElectroMax DH10 B Escherichia coli competent cells (Invitrogen).
8
Multi-Omics Integration of Brain Transcriptome and Epigenome
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In ROSMAP, there were 421 subjects with both data of DNA methylation and RNA sequencing (RNA-seq) (Illumina) from their dorsolateral prefrontal cortex 9 (Supplementary Methods). We included the 17,068 autosomal genes in the unit of normalized log2(cpm) into the transcriptome-wide association study (TWAS). A subset of these subjects (N=413) were previously used to derive the 47 cell-type relevant co-expressed gene module 23 .
In MSBB, we downloaded the gene expressions of the transcriptome from Synapse platform (https://www.synapse.org/#!Synapse:syn7391833). Based on the genotype concordance check (Supplementary Methods), we included 50 subjects who have been profiled with both DNA methylation at prefrontal cortex and RNA-seq (Illumina) at BM44 region (closest to the prefrontal cortex).
In MAYO, we included 45 AD cases with both the DNA methylation data at cg05157625 and microarray-based gene expression data from their temporal cortex (Illumina) 12, 13 .
In MSBB, we downloaded the gene expressions of the transcriptome from Synapse platform (https://www.synapse.org/#!Synapse:syn7391833). Based on the genotype concordance check (Supplementary Methods), we included 50 subjects who have been profiled with both DNA methylation at prefrontal cortex and RNA-seq (Illumina) at BM44 region (closest to the prefrontal cortex).
In MAYO, we included 45 AD cases with both the DNA methylation data at cg05157625 and microarray-based gene expression data from their temporal cortex (Illumina) 12, 13 .
9
Comparative Transcriptomic Analysis of Starch Biosynthesis
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Transcriptome data for starch biosynthetic genes for Brachypodium and barley were extracted from previously published data sets obtained from two independent studies: Davidson (2012) (link) and Radchuk (2010), respectively. A Brachypodium gene expression matrix was generated using RNA-Seq Illumina technology, while barley mRNA expression profiles were collected in a cDNA macroarray dataset. Data from barley were unlogged and transformed to relative values (fold change) to allow identification of expression values, and expression trends for starch-related genes in both species were analysed individually.
10
Transcriptional Profiling of Fungal Strain Mutants
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Fresh 100 µL conidia (1 × 108/mL) from the WT and ∆Focpmi1 strains were cultured in YPD medium, supplemented with 2 mM mannose for the mutant. The cultures were incubated at 28 °C at 200 rpm for 48 h. After 2 days of incubation, mycelia were filtered, washed twice with autoclaved water, dried well, and freeze-stored in liquid nitrogen. RNA isolation and sequencing were conducted by Biomarker Technologies (China). High-quality RNA samples were prepared and sequencing was performed on the Illumina sequencing platform. The differentially expressed genes (DEGs) were selected using DESeq2 with Fold Change ≥ 5 and FDR < 0.01. DEGs were visualized using a volcano plot and COG analysis was performed to characterize the biological functions of the DEGs and the metabolic pathways in which they are involved. The RNA-Seq Illumina reads were deposited in the National Center for Biotechnology Information Short Read Archive (NCBI-SRA) and are publicly available under the accession number PRJNA940601.
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