1μg of total RNA was used for mRNA- and miRNA-seq. For mRNA-seq polyA-selected mRNAs were processed for unstranded libraries using the TruSeq v2 kit (Illumina) that underwent 50bp paired-end sequencing on an Illumina HiSeq 2500 machine. Paired-end reads were mapped to the reference mouse NCBIM37 genome using Tophat2 and Ensembl release 67 annotations (May 2012 data freeze). Count level data was quantified using union gene models with HTseq-counts. For miRNA sequencing a second sample from the same total RNA fraction was used and libraries were prepared using Truseq small-RNA library prep kit (Illumina). The 50bp single-end reads were processed for miRNA expression using the miRDeep2 analysis pipeline. For additional information on sequencing, read alignment parameters and data-preprocessing, please see the Online Methods .
Truseq v2 kit
Manufactured by Illumina
Sourced in United States
The TruSeq v2 kit is a laboratory equipment product developed by Illumina. It is designed to prepare DNA samples for sequencing on Illumina platforms. The kit provides reagents and protocols for library preparation, which is a crucial step in the next-generation sequencing workflow.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (8 mentions)
Rneasy kit, by Qiagen (7 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Hiseq 2000, by Illumina (4 mentions)
Bioanalyzer, by Agilent Technologies (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Truseq small rna kit, by Illumina (3 mentions)
Truseq small rna library prep kit, by Illumina (2 mentions)
Hiseq 2500 system, by Illumina (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Trizol rna extraction reagent, by Thermo Fisher Scientific (2 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
Uracil dna glycosylase, by Thermo Fisher Scientific (1 mentions)
Rneasy 96 kit, by Qiagen (1 mentions)
Total rna isolation system, by Promega (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Rna extraction kit, by Qiagen (1 mentions)
Direct zol rna kit, by Zymo Research (1 mentions)
38 protocols using truseq v2 kit
1
Comprehensive Transcriptome Profiling of Mouse
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Total RNA Extraction and Strand-Specific RNA-Seq
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We extracted total RNA using RNeasy 96 kits (Qiagen, Valencia, CA) based on the manufacturer’s guideline (Protocol for Isolation of Total RNA from Animal Cells using spin technology, Cat#19504). We prepared PolyA+ RNA-Seq libraries as in described previously82 (link). We added spike-in RNA from the External RNA Control Consortium (ERCC, Baker et al.)83 (link) during the RNA fragmentation step. For RNAi control samples (dsRNA for LacZ), we added ERCC pool 78A. For the rest of the samples, we added ERCC pool 78B. They are from the National Institute of Standards and Technology (Standard Reference Material 237484 (link)). We incorporate dUTP during the second strand synthesis of the reverse transcription step in order to generate strand-specific libraries. We ligated 24 different indexed adapters from TruSeq v2 kit (Illumina, San Diego, CA) to reverse-transcribed cDNA for multiplexed sequencing. We enzymatically removed dUTP-incorporated strands from the ligation outcome before PCR using Uracil-DNA glycosylase (Thermo Fisher Scientific, Waltham, MA). Sequencing was done with Illumina HiSeq 2500 (Illumina, San Diego, CA) as 50 bp single-end sequencing at the NIDDK Genomics Core (Bethesda, MD).
3
Transcriptome Sequencing of Radial Nerve Cords in Asterias rubens
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Radial nerve cords (approx. 30 mg) dissected from a male adult specimen of A. rubens were used for RNA isolation (Total RNA Isolation System, Promega). Library preparation (TruSeqv2 kit, Illumina) was performed at the QMUL Genome Centre and sequencing was performed on an Illumina HiSeq platform at NIMR (Mill Hill), with cBot used to generate clusters. A total of 168 776 495 × 100 bp reads were obtained and raw sequence data (SRP068147; http://www.ncbi.nlm.nih.gov/sra/SRP068147 ) were assembled using SOAPdenovo -Trans v. 1.0 (http://soap.genomics.org.cn/SOAPdenovo-Trans.html ), a short-read assembly method developed by the Beijing Genomics Institute [35 (link)]. Contigs were assembled from reads with an overlap greater than 31 bp, which were then mapped back to the raw reads. The 326 816 contigs generated (with 16 316 over 1000 bp) were then set up for BLAST analysis using Sequence Server , which is freely available to academic users (http://www.sequenceserver.com ) [36 ].
4
Transcriptome Analysis of DH Onion
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Seed lots of the long day DH bulb onion ‘CUDH2107’ (line CUDH066607 in [11 ]) were provided by Cornell University (US). Total RNA was extracted from tissue samples pooled from multiple plants grown in tunnel houses at Lincoln New Zealand (latitude 42° S) or in controlled environments. The stages sampled were as follows: leaves (from plants grown in long day of 16 h light: 8 h dark), floral buds from unexpanded umbels, unopened florets from expanded umbels, open florets with pollen, older flowers and roots. RNA was isolated using a Qiagen RNA extraction kit following the manufacturer’s guidelines. Libraries were made using the TruSeq v2 kit (Illumina), and were sequenced on the Illumina HiSeq 2000 platform by NZGL Ltd.
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Transcriptome Analysis of Mouse RNA-seq
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Hippocampal RNA Sequencing in Rat ELA
7
Transcriptomic Analysis of Cerebral Palsy
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RNA-Seq data corresponding to 182/186 CP cases in this study and 20 unrelated control samples were generated as part of a separate and dedicated gene expression analysis study for CP.15 (link) We incorporated this expression data resource into a new analysis to determine the influence of CNV identified in this study on gene expression. Briefly, RNA was extracted from Epstein Barr Virus transformed LCL using QIAGEN RNeasy kits, as per the manufacturer’s protocol. Library preparation and RNA-Seq were performed as a service by the UCLA Neuroscience Genomics Core Facility. The TruSeq v2 kit (Illumina) was used to generate un-stranded libraries with 150 bp mean fragment sizes and 50 bp pair end sequencing performed using the HiSeq2500 (Illumina). Sequence data were mapped to Illumina iGenomes hg19 build of the human genome using tophat2.33 (link) Read counts were generated with htseq-count.34 (link) Z-statistics for each gene in each sample were calculated over all 202 samples using the scale function from the R statistical programming language v3.4.4.
8
RNA Extraction and Sequencing Protocol
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Frozen root samples were homogenized by grinding to a fine powder in liquid nitrogen. Total RNA was extracted with Trizol reagent from approximately 50 mg as per the manufacturer’s instructions. Total RNA was precipitated by centrifugation at 12,000 g for 15 min at 4 . The resulting pellet was washed three times with 75 % (v/v) ethanol before being resuspended in 40 L of DEPC−treated water heated to 65 .
RNA samples with RNA integrity number (RIN) < 5 (Agilent 2100 Bioanalyzer) were discarded and new extractions conducted. RNA-seq libraries were constructed using the Illumina TruSeq v2 kit following the manufacturer’s published protocol [87 (link)], pooled, and sequenced on an Illumina Nextseq 500 instrument with a target read length of 2x75 nucleotides and a target sequencing depth of 20 M paired-end reads per sample.
RNA samples with RNA integrity number (RIN) < 5 (Agilent 2100 Bioanalyzer) were discarded and new extractions conducted. RNA-seq libraries were constructed using the Illumina TruSeq v2 kit following the manufacturer’s published protocol [87 (link)], pooled, and sequenced on an Illumina Nextseq 500 instrument with a target read length of 2x75 nucleotides and a target sequencing depth of 20 M paired-end reads per sample.
9
Transcriptomic Profiling of iPSC-derived Cardiomyocytes
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Total RNA was extracted from flash-frozen pellets of 1 million cells using TRI Reagent (Sigma #T9424) and a homogenizer followed by RNA isolation and clean-up using the Direct-zol RNA Kit (Zymo Research #11–331). RNA-seq libraries were generated with the Illumina TruSeq V2 kit (Illumina, RS-122–2001) and 1 μg of RNA, following manufacturer’s instructions. Libraries were made from RNA isolated from three independent iPSC-CM differentiations (triplicates of iPSC and of cardiomyocytes). Libraries were sequenced on an Illumina HiSeq 4000.
Gene counts were quantified with Salmon 0.7.2 (Patro et al., 2017 (link)) and imported with tximport 1.2.0 (Soneson et al., 2015 (link)) into DESeq2 1.12.4 (Love et al., 2014 (link)) to call differentially expressed genes. A minimum 1.5-fold-difference between CMs and iPSC triplicates and a minimum adjusted p-value of 0.05 were required to select differentially expressed genes for downstream analyses. TPMs (transcripts per million) were also estimated by Salmon. Because the samples clearly clustered according to their known tissues of origin (Figure 1—figure supplement 2A ), no correction for batch effects was performed.
Gene counts were quantified with Salmon 0.7.2 (Patro et al., 2017 (link)) and imported with tximport 1.2.0 (Soneson et al., 2015 (link)) into DESeq2 1.12.4 (Love et al., 2014 (link)) to call differentially expressed genes. A minimum 1.5-fold-difference between CMs and iPSC triplicates and a minimum adjusted p-value of 0.05 were required to select differentially expressed genes for downstream analyses. TPMs (transcripts per million) were also estimated by Salmon. Because the samples clearly clustered according to their known tissues of origin (
10
Bulk RNA-seq Library Preparation and Analysis
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