Total RNA was isolated by the guanidine thiocyanate (GTC) method using standard protocols54 (link). PolyA+ RNA fraction was processed as in Illumina’s TruSeq RNA Sample Preparation v2 Protocol. The resulting purified complementary DNA (cDNA) library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer’s protocols. RNA-seq data sets were analyzed using the tool Nextpresso55 . Nextpresso is comprises four basic levels: 1. Quality check, 2. Read cleaning and/or down-sampling, 3. alignment, and 4. analysis (gene/isoform expression quantification, differential expression, gene-set enrichment analysis and fusion prediction). Gene signatures (Hallmark genesets) were downloaded from GSEA—Molecular Signature Database for Gene set enrichment analysis.
Truseq cluster generation kit v5
Manufactured by Illumina
Sourced in United States
The TruSeq cluster generation kit v5 is a laboratory equipment product used for preparing DNA samples for sequencing. It performs the cluster generation step, which is a crucial part of the Illumina sequencing workflow. The kit contains reagents and materials necessary to generate DNA clusters on the flow cell surface, which is required for Illumina sequencing platforms.
Automatically generated - may contain errors
Lab products found in correlation
Flow cell, by Illumina (5 mentions)
Genome analyzer iix, by Illumina (5 mentions)
Truseq rna sample preparation v2, by Illumina (2 mentions)
Truseq rna sample preparation v2 protocol, by Illumina (2 mentions)
Truseq dna sample preparation guide, by Illumina (1 mentions)
Pe primers, by Illumina (1 mentions)
Ercc exfold rna spike in mixes, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna sample preparation part, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Truseq rna sample preparation guide, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
8 protocols using truseq cluster generation kit v5
1
RNA-Seq Protocol for Transcriptome Analysis
2
ChIP-seq Library Preparation Protocol
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ChIP was performed as described above. DNA (20 ng) was quantified by fluorimetry, resolved by electrophoresis and fractions of 50–250 bp were extracted. Input samples correspond to balanced blends of inputs from selected samples. Fractions were processed through subsequent enzymatic treatments of end repair, dA tailing and ligation to adaptors following Illumina's ‘TruSeq DNA Sample Preparation Guide' (part #15005180 Rev. C). Adaptor-ligated libraries were amplified by limited-cycle PCR with Illumina PE primers (12 cycles). The resulting purified DNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents following the manufacturer's protocols.
3
Transcriptome Analysis by RNA-seq
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Total RNA was isolated by the guanidine thiocyanate (GTC; VWR AMRESCO Chemicals, Cat no. K965-250ML) method using standard protocols [29 (link)]. PolyA+ RNA fraction was processed as in Illumina’s ‘‘TruSeq RNA Sample Preparation v2 Protocol’’. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer’s protocols. RNA-seq data sets were analyzed using the tool Nextpresso [30 (link)]. Nextpresso is comprised of four basic levels: 1. Quality check, 2. Read cleaning and/or down-sampling, 3. Alignment, and 4. Analysis (gene / isoform expression quantification, differential expression, gene set enrichment analysis and fusion prediction. Gene signatures (Hallmark gene sets) were downloaded from GSEA - Molecular Signature Database for Gene set enrichment analysis. Data deposited in the NCBI SRA database (Accession: PRJNA832709).
4
ChIP-seq Library Preparation Protocol
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ChIP was performed as described above. DNA (15 ng) was quantified by fluorimetry, electrophoresis was resolved and factions of 50–300 bp were extracted. DNA was blunted and phosphorylated with T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. An A nucleotide was then added to the 3′ end of the DNA fragments. Adaptor-ligated libraries were amplified by 20 cycles of PCR amplification. Purified DNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and subjected to massively parallel sequencing (Illumina Genome Analyzer IIx) following the manufacturer's protocols.
5
Transcriptome Profiling by Illumina Sequencing
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Total RNA (1 μg) was spiked with ERCC ExFold RNA spike-In mixes (Life Technologies). RNA quality was assessed on an Agilent 2100 Bioanalyzer and samples with a RNA integrity number >8.5 were used. PolyA+ fractions were purified, randomly fragmented, converted to double-stranded cDNA and processed through subsequent enzymatic treatments of end repair, dA tailing and ligation to adaptors following Illumina ‘TruSeq Stranded mRNA Sample Preparation Part # 15031047 Rev. D' (this kit incorporates dUTP during second-strand cDNA synthesis, which implies that only the cDNA strand generated during first-strand synthesis is eventually sequenced). Adaptor-ligated libraries were generated by PCR with Illumina PE primers (eight cycles). The resulting purified cDNA libraries were applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following the manufacturer's protocols.
6
TruSeq RNA Sample Preparation v2 Protocol
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Library construction was carried out following the TruSeq® RNA Sample Preparation v2 (Illumina Inc, USA) protocol. RNA-Seq was performed using the TruSeq Cluster Generation Kit v5 (Illumina Inc, USA), following the manufacturer’s instructions. The six libraries were distributed into 2 lanes and underwent paired-end sequencing (PE 2 × 101 bp) using the HISEQ2500 Illumina Platform through High Output run.
7
Transcriptome Sequencing of Mouse Embryonic Fibroblasts
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Asynchronous MEFs (3 clones) were harvested and RNA was extracted using RNeasy kit from Qiagen. PolyA + RNA was purified with the Dynabeads mRNA purification kit (Invitrogen), randomly fragmented and converted to double-stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing and ligation to adapters as in Illumina’s ‘TruSeq RNA Sample Preparation Guide’ (Part # 15031047 Rev. D). Adapter-ligated library was completed by limited-cycle PCR with Illumina PE primers and applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on HiSeq2000 following manufacturer’s protocols. Fastq files with 86-nt single-end sequenced reads were quality-checked with FastQC (S. Andrews, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) and aligned to the mouse genome with Nextpresso executing TopHat-2.0.0 using Bowtie 0.12.7 and Samtools 0.1.16 allowing two mismatches and five multi-hits [64 (link)]. Reads were mapped to mm10 genes using HTSeq [65 (link)].
8
RNA-Seq Transcriptome Analysis Protocol
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Total RNA was isolated by the guanidine thiocyanate method using standard protocols. PolyA+ RNA fraction was processed as in Illumina's ''TruSeq RNA Sample Preparation v2 Protocol'' (Part # 15026494 Rev. C). The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer's protocols. Transcript assembly and estimation of their abundances were calculated with Cufflinks 1.3.0 (Trapnell et al., 2012) , using the human genome annotation dataset Homo_sapiens.GRCh37.65 from Ensembl (Flicek et al., 2014) . Differential expression for genes across the different conditions was calculated with Cuffdiff (Trapnell et al., 2012) . Principal component analysis (PCA) figures showing how samples clustered was obtained with R (http://www.r-project.org/). Heatmaps showing gene expression levels (in FPKM, fragments per kilobase of transcript per million mapped fragments) through the different samples were drawn with GENE-E (http://www.broadinstitute.org/cancer/software/ GENE-E/) for a subset of selected genes. Data deposited in ArrayExpress: E-MTAB-3808. More details are available in the Supplemental Information.
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