Transcriptomic analysis was used to determine the gene expression profile of distal intestine and hepatic RNA (n = 3) in adult fish from all groups, including the negative control (FM group), the positive control (SBM group), and the tested drug inclusion group (SB group). The process for preparing the gene library and sequencing the transcriptome was consistent with previously described methods (Johnson and Hofmann, 2016 (link)). To summarize, sequencing libraries were prepared using the NEBNext RUltraTM RNA Library Prep Kit for Illumina R (NEB, USA) according to the manufacturer’s guidelines, and the quality of the libraries was determined using the Agilent Bioanalyzer 2100 system. On an Illumina platform (NovaSeq 6000), the library preparations were sequenced and 150 bp paired-end reads were produced.
Nebnext r ultra rna library prep kit for r
Manufactured by Illumina
Sourced in United States
The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® is a library preparation kit designed for the generation of high-quality sequencing libraries from RNA samples. The kit provides a streamlined workflow for the conversion of RNA into adaptor-ligated cDNA fragments, which can then be sequenced on Illumina platforms.
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Lab products found in correlation
Novaseq 6000, by Illumina (3 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Hiseq platform, by Illumina (2 mentions)
Ampure xp system, by Beckman Coulter (1 mentions)
Nanophotometer r spectrophotometer, by Implen (1 mentions)
Qubit r 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Transzol up plus rna kit, by Transgene (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Agilent Technologies (1 mentions)
6 protocols using nebnext r ultra rna library prep kit for r
1
Transcriptomic Analysis of Distal Intestine and Liver
2
Transcriptomic Analysis of Fish Hindgut
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For the hindgut’s RNA (n = 6), transcriptomic analysis was completed to reveal the gene expression profile systematically in adult fish from all groups, including the negative control (FM group), the positive control (SBM group), and the tested drug inclusion group (SN group). The procedure of the gene library preparation and sequencing for transcriptome followed previously published methods (46 (link)). Briefly, sequencing libraries were generated using NEBNext R UltraTM RNA Library Prep Kit for Illumina R (NEB, USA) following the manufacturer’s recommendations, and the library quality was assessed on the Agilent Bioanalyzer 2100 system. The library preparations were sequenced on an Illumina platform (NovaSeq 6000), and 150 bp paired-end reads were generated.
3
RNA-seq Library Preparation Protocol
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RNA samples from 3 biological replicates were purified using poly-T oligo-attached magnetic beads. Then, sequencing libraries were conducted using NEBNext R UltraTM RNA Library Prep Kit for Illumina R (NEB) according to the manufacturer's instructions. The library fragments were purified with AMPure XP system (Beckman Coulter, Brea, CA, USA) for cDNA fragments of preferentially 250-300 bp in length. The library preparations were sequenced on an Illumina Hiseq platform, and 125 bp/150 bp paired-end reads were generated with quality assessed.
4
RNA-Seq Transcriptome Assembly and Annotation
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First, we assessed the quality and quantity of RNA samples by using agarose gels, the NanoPhotometer R Spectrophotometer (IMPLEN, CA, United States), the Qubit R 2.0 Fluorometer (Life Technologies, CA, United States), and the Bioanalyzer 2100 System (Agilent Technologies, CA, United States), respectively. Next, 1 µg of total RNA for each tissue evenly mixed by three biological pools was used to constructed a cDNA library using the NEBNext R Ultra TM RNA Library Prep Kit for Illumina R (NEB Inc., United States). Lastly, the prepared libraries were sequenced with 150-bp paired-end reads on an Illumina HiSeq TM 2000 instrument.
After cDNA libraries were sequenced, redundant sequences were discarded from the resulting raw reads, including adapter, low quality and containing poly-N sequences. The qualityfiltered reads (clean reads) were assembled into transcripts by Trinity v2.5.1 (Grabherr et al., 2011) (link) as the transcript transcriptome. Further, the assembled transcripts were clustered by Corset v1.05 (Davidson and Oshlack, 2014) (link), and the longest transcript for each cluster was selected as one unigene. All the unigenes were pooled into the unigene transcriptome.
After cDNA libraries were sequenced, redundant sequences were discarded from the resulting raw reads, including adapter, low quality and containing poly-N sequences. The qualityfiltered reads (clean reads) were assembled into transcripts by Trinity v2.5.1 (Grabherr et al., 2011) (link) as the transcript transcriptome. Further, the assembled transcripts were clustered by Corset v1.05 (Davidson and Oshlack, 2014) (link), and the longest transcript for each cluster was selected as one unigene. All the unigenes were pooled into the unigene transcriptome.
5
Transcriptome Analysis of Crustacean Ganglia
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Crabs were chilled on ice and dissected for thoracic ganglia. For every group, samples were collected in three different pools; each pool contained five biological replicates. Total RNA was prepared using a column-based TransZol Up Plus RNA kit (TransGen Biotech, China). The concentration of the extractive RNA was determined using the Thermo Fisher Scientific Nanodrop 2000 (Waltham, MA, USA), and the quality and integrity were determined with an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) and a Qubit 2.0 Fluorometer (Life Technologies, CA, USA). The qualified RNA samples were used for cDNA library construction. Sequencing was done with the NEBNext R Ultra™ RNA Library Prep Kit for Illumina R (NEB, Ipswich, MA, USA), and index codes were added to tag these sequences. The libraries were sequenced using the Illumina HiSeq platform.
6
Transcriptomic Analysis of Intestinal RNA
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Transcriptomic analysis was used to systemically disclose the expression of intestinal RNA (n = 3) in both the intestinal tissue (IT group) and current prepared intestinal mucosa cell suspension (IMC group). The process for preparing the gene library and sequencing the transcriptome was done according to previously described methods (41 (link)). In brief, sequencing libraries were prepared using the NEBNextRUltra™ RNA Library Prep Kit for Illumina R (NEB, USA), and the quality of the libraries was determined using the Agilent Bioanalyzer 2100 system. On an Illumina platform (NovaSeq 6000), the library preparations were sequenced and 150bp paired-end reads were produced.
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