Total RNA and DNA were isolated from the same cell samples. The DNA was used to normalize the relative cell number in different samples by qPCR. Reverse transcription was performed with 1 µg of total RNA using the first-strand cDNA synthesis kit with random hexamers (0.2 µg per reaction) according to the manufacturer's instructions (GE Healthcare). All primer sequences used are given in Supplemental Table S1. For whole-transcriptome analysis, total RNA was depleted of ribosomal sequences with the RiboZero kit (Epicentre), and sequencing libraries were generated with the ovation RNA-seq system V2 kit or encore complete RNA-seq library system (NuGEN) and paired-end sequencing with Illumina HiSeq. For more details, see the Supplemental Methods.
Encore complete rna seq library system
Manufactured by Tecan
Sourced in United States
The Encore Complete RNA-Seq Library System is a laboratory equipment product that enables the preparation of high-quality RNA-Seq libraries from total RNA samples. The system provides a comprehensive workflow for library construction, including steps for RNA fragmentation, cDNA synthesis, adapter ligation, and library amplification.
Automatically generated - may contain errors
Lab products found in correlation
Ribo zero kit, by Illumina (1 mentions)
Ovation rna seq system v2 kit, by Tecan (1 mentions)
First strand cdna synthesis kit, by GE Healthcare (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
Nd 1000, by Agilent Technologies (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Bioanalyzer 2200, by Agilent Technologies (1 mentions)
Nucleospin rna, by Macherey-Nagel (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer pico chips, by Agilent Technologies (1 mentions)
7 protocols using encore complete rna seq library system
1
Transcriptome Analysis via RNA-seq
2
Targeted RNA-Seq Transcript Detection
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Example 4
An RNA sequencing library is made from NuGEN's Encore Complete RNA-Seq Library System. The library is sequenced on an Illumina DNA sequencer. Following the first sequencing read, a pool of primers that will hybridize to specific exons of interest is injected into the sequencing machine. These primers are used to generate a second sequencing read in a downstream exon. The second, targeted sequencing read provides information about which exons have been spliced together to generate a particular RNA transcript.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments provided herein described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
3
Transcriptomic Analysis of MIDY Porcine Liver
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Liver samples were homogenized in Trizol, and total RNA was isolated with chloroform following manufacturer's protocol. Isolated total RNA was quantified (Nanodrop, ND1000) and quality controlled (Agilent, Bioanalyzer 2100). Good quality RNA (RIN >7.0) was used to construct sequencing libraries (Nugen, Encore Complete RNA-Seq library system). This kit enables the analysis of transcriptome profiles with reduced representation of ribosomal RNA because of not so random priming during cDNA synthesis. All libraries were sequenced on a HiSeq 1500 (Illumina) as 100 b single reads. Demultiplexing and quality control were performed on the obtained FastQ files followed by mapping to the S.scrofa 11.1 reference genome using the gapped-mapper STAR. HTSeq [12] using strict intersection mode and a minimum alignment quality of 10 was used to quantify the number of hits to each gene. DESeq2 [13] (link) with outlier replacement and independent filtering was used to detect differentially abundant transcripts between MIDY and WT samples. Pre-ranked unweighted gene set enrichment analyses (GSEA) [14] (link) were performed on the signed log transformed p-values as ranking metric using MSigDB [15] and the specific Sus scrofa KEGG pathways [16] (link). For network visualization, Cytoscape [17] (link) was used with the ClueGO [18] (link) and CluePedia apps to analyze significant genes.
4
RNA-Seq Library Generation and Targeted Sequencing
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Example 4
An RNA sequencing library is made from NuGEN's Encore Complete RNA-Seq Library System. The library is sequenced on an Illumina DNA sequencer. Following the first sequencing read, a pool of primers that will hybridize to specific exons of interest is injected into the sequencing machine. These primers are used to generate a second sequencing read in a downstream exon. The second, targeted sequencing read provides information about which exons have been spliced together to generate a particular RNA transcript.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
5
Hypoxia-Induced Transcriptional Profiling
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RNA was extracted and pooled from 3 independent 3D-cultures grown under hypoxic or normoxic conditions. Ribosomal RNA in the pooled RNA was depleted using Encore Complete RNA-Seq Library Systems (NuGEN, San Carlos, CA, USA) and RNA was sequenced using an Illumina HiSeq 2000 sequencer (San Diego, CA, USA) at the Functional Genomics Center Zurich. Isoform and gene expression levels were computed with RSEM (Version 1.2.0; http://www.biomedcentral.com/1471-2105/12/323 ). RSEM was run in stranded mode with the additional option to estimate the distribution of the read start positions. The expression levels (RSEM's posterior estimate of the read counts per transcript normalized for gene length) were normalized across samples using the geometric mean of all expression signals. To attenuate expression ratios for low abundance genes, a fixed value of 5 was added to all expression values before computing the log-ratio. Threshold levels for hypoxia- or LPS-dependent up- or down-regulation were set at >1.5 or <0.67, respectively, as were the threshold levels for HIF-1α or p65/RelA dependency of up- or down-regulated gene expression when compared to the respective shMOCK controls. Only genes with a minimal expression level of 10 were considered biologically relevant. All data were deposited in the Short Read Archive (SRA) of the NCBI and are accessible through the ID SRP043151.
6
RNA-Seq Library Preparation Using Encore
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After isolation of RNA, the Encore Complete RNA-Seq Library Systems (NuGen) protocol was followed using 500 ng of RNA per sample as starting material. Half of the ligated reaction volume was used for PCR (14 cycles) and the other half was kept at −80°C as a backup. Libraries were checked for quality and quantified using the Bioanalyzer 2200 (Agilent, Santa Clara, CA), before being sequenced in barcoded pools on the Illumina Hiseq 2000 instrument (50 single sequencing, 3 lanes; Sequencing Core, UMICH).
7
Transcriptional Profiling of Drosophila Ring Glands
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We carefully staged control (phm>w1118) and phm>séan-RNAi (VDRC #100854) larvae at the L2/L3 molt, and we dissected ring glands at 44 ± 0.5 hr after the molt. These samples were then transferred to ice-cold TRIzol reagent (Thermo Fisher Scientific). For each sample, the lysates of 20 ring glands were vortexed at room temperature for 5 sec, briefly spun down, and stored at −80° until use. Total RNA was isolated by NucleoSpin RNA (Macherey-Nagel, Düren Germany), quantified by RiboGreen Quanti Kit (Thermo Fisher Scientific), and RNA integrity was analyzed by Agilent Bioanalyzer Pico chips. We used 100 ng of total RNA per sample as input for cDNA library synthesis. Each genotype was analyzed by two independent biological replicates for analysis. The Encore Complete RNA-Seq library systems (NuGEN Technologies, San Carlos, CA) were used to produce the cDNA libraries for next-generation sequencing, following the manufacturer’s instructions. The cDNA libraries resulting from the Encore RNA-Seq systems were pooled together in equal concentrations for sequencing at McGill University and the Génome Québec Innovation Centre (Montréal, Canada). We normalized raw data with ArrayStar 4.0 (DNASTAR), and data were analyzed by ArrayStar 4.0, Access (Microsoft, Redmond, WA), and the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang et al. 2009 (link)).
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