Transcriptome sequencing was conducted on muscle samples obtained 40 days after SsMeox1 knockdown with 3 knockdown samples and 3 control samples. RNA extraction, sequencing library construction, and Illumina sequencing were performed by Novogene Bioinformatics Technology Co., Ltd. Briefly, the total RNA of the six samples (three biological replications per group) was extracted using Invitrogen TRIzol (ThermoFisher, Waltham, MA, USA). The RNA concentration and quality were analyzed using a Fragment Analyzer 5400 (Agilent, Santa Clara, CA, USA). The NEBNext® UltraTM RNA Library Prep Kit (NEB, Ipswich, MA, USA) was utilized for library preparation. The library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). These libraries were sequenced using the NovaSeq 6000 platform. Salmon version 0.7.2 was used for the quantification of the transcript counts [48 (link)]. The gene expression levels were standardized by the transcripts per kilobase million (TPM) [49 (link)]. The analysis of differentially expressed genes (DEGs) was performed using the R package DESeq2 [50 (link)], and the genes with |log2 FC| ≥ 1 and an adjusted p-value < 0.01 were assigned as DEGs. In addition, GO and KEGG enrichment analysis were performed by DAVID (https://david.ncifcrf.gov/ (accessed on 14 October 2021)), and the results were visualized by R Studio.
Nebnext ultratm rna library prep kit
Manufactured by New England Biolabs
Sourced in United States, China
The NEBNext® UltraTM RNA Library Prep Kit is a laboratory tool designed for the preparation of RNA libraries for next-generation sequencing. It provides a streamlined workflow for converting RNA into cDNA libraries.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (10 mentions)
Hiseq platform, by Illumina (9 mentions)
Novaseq platform, by Illumina (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Novaseq 6000, by Illumina (5 mentions)
Hiseq 2500, by Illumina (5 mentions)
Nanophotometer spectrophotometer, by Implen (4 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (4 mentions)
Hiseq x ten platform, by Illumina (4 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (4 mentions)
Hiseq 2000, by Illumina (3 mentions)
Hiseq 2500 platform, by Illumina (3 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (3 mentions)
Novaseq 6000 platform, by Illumina (3 mentions)
Hiseq 4000 platform, by Illumina (3 mentions)
Hiseq 2000 platform, by Illumina (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Random hexamer primers, by Illumina (2 mentions)
74 protocols using nebnext ultratm rna library prep kit
1
Transcriptomic Analysis of SsMeox1 Knockdown
2
Transcriptomic Analysis of H. pylori Heat Stress Response
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H. pylori NCTC 11637 strain was divided into two groups in triplicate. The experimental group was treated at 41°C for 3 days. The control group was treated at 37°C for 3 days. The cells were harvested, and total RNA was extracted and purified using the Bacterial RNA kit (Omega Bio-tek, GA, United States) according to manufacturer protocols. Quality control of each RNA sample was performed with Agilent 2100 Bioanalyzer (Agilent Technologies, Beijing, China). The cDNA libraries were constructed using NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs, Ipswich, MA, United States) and submitted for sequencing using IlluminaHiseq 4000. Library construction and sequencing were performed by Allwegene BioTech Co., Ltd. (Beijing, China). Raw reads were filtered using Trimmomatic v0.33 (Bolger et al., 2014 (link)) and mapped to the H. pylori NCTC 11637 genome (National Center for Biotechnology Information [NCBI], 2012 ) using Bowtie2 v2.2.6 (Langmead and Salzberg, 2012 (link)) with default parameters. Genes were quantified using HTSeq v0.6.0 (Anders et al., 2015 (link)). Differentially expressed genes (DEGs) were analyzed by DESeq2 v1.22.1 in R (Love et al., 2014 (link)). Genes with absolute log2 fold-changes > 1 and multiple testing adjusted (Benjamini–Hochberg procedure) q < 0.05 were considered as DEGs.
3
Testicular Transcriptional Profiling at Postnatal Day 35
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Total RNA was isolated from whole testes at 35 days postpartum (P35). RNA‐seq libraries were constructed from 1 μg of RNA using a NEBNext UltraTM RNA Library Prep Kit (NEB, USA). Sequencing was performed on an Illumina NovaSeq 6000 platform with 350‐bp paired‐end reads. Differentially expressed gene (DEG) analysis was performed using the DESeq2 R package. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was implemented with the clusterProfiler R package. Library construction, sequencing, and data analyses were performed by Annoroad Genomics Co., Ltd. (Beijing, China).
4
RNA-Seq Protocol for Differential Gene Expression
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After total RNA was extracted, mRNA was isolated by Oligo Magnetic Beads and cut into small fragments for cDNA synthesis. Libraries were generated using the NEBNext UltraTM RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) for the Illumina system following the manufacturer’s instructions. Sequencing was conducted using the Illumina HiSeq XTEN platform. The mRNAs with p < 0.05 between GC and paired normal samples were identified to be differentially regulated. The detailed data processing procedure was presented in Supplementary Material S2 .
5
RNA-Seq Library Preparation and Sequencing
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About 30 μg RNA from the stress-treated samples and normal-cultured samples was mixed together and used to construct the transcriptome sequencing library. The mRNA was first purified from the total RNA by using Dynabeads® Oligo (dT)25 (Invitrogen 610–05, USA) and then fragmented. Double-stranded cDNA was synthesized using transcriptase and random hexamer primers. The sequencing library was constructed using the NEBNext® UltraTM RNA Library Prep Kit (NEB, USA) and then loaded onto an Illumina HiSeq2000 platform for PE 2 × 100 bp sequencing. In all, 35,534,655 paired-end reads were obtained and used for the following analysis. The generated raw sequencing data was available in the Sequence Read Archive (BioProject ID: PRJNA404009), NCBI.
Raw reads were first subjected to preliminary processing by using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/ ) to remove reads that contained adapter sequences, poly-N sequences (greater than 10%), low-quality reads, and reads with lengths less than 40 bp. The clean reads were assembled using Trinity v2013-02-25 by using the default parameters (http://trinityrnaseq.sourceforge.net/ ). Redundant transcripts were avoided by extracting the longest transcript of each gene as a unigene for downstream analysis.
Raw reads were first subjected to preliminary processing by using the FASTX-Toolkit (
6
Transcriptomics of Wound Healing in Seaweed
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Three replicates of SAHA-treated fragments were sampled at the 6th hour, 1, 2, 3, and 5 days after wounding by collection and centrifugation. Triplicates of “uncut thalli” were collected as controls. RNA isolation, mRNA-Seq library preparation were done with NEBNext® UltraTM RNA Library Prep Kit (NEB, America) following the manufacture’s instruction. PE 2×100 bp sequencing was performed on Illumina HiSeq2000 platform for each sample. After filtering out low quality reads, adapter sequences and reads containing N, the generated clean data were aligned to the assembly genome using HISAT2 (Kim et al., 2019 (link)). The mRNAs expression was calculated as Reads Per Kilobase per Million reads (RPKM). The differential expression of mRNAs was performed using the DESeq2 R package (1.10.1) (Love et al., 2014 (link)). The criterion of adjusted P-value <0.05 was used to identify differentially expressed genes.
7
RNA-Seq analysis of L. monocytogenes stress response
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Approximately 1 × 106 CFU of L. monocytogenes was incubated in BHI and BHI supplemented with MEL-A (32 μg/mL) and LA (2 mg/mL) at 37 °C for 12 h. Bacterial cells were then collected by centrifugation at 2500× g for 5 min and then snap-frozen in liquid nitrogen. Next, total RNA was extracted using the method described by Kafantaris [26 (link)]. Then, total RNA from all samples was sent to Novogene company (Beijing, China) for subsequent library construction and transcription sequencing. Briefly, the libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs, Los Angeles, CA, USA) according to the manufacturer’s instructions. Then, these libraries were sequenced on the Illumina Novaseq platform (Illumina, San Diego, CA, USA) and 150 bp paired-end readings were generated. Three biological replicates were performed in both the experimental group and the control group. Next, in-house perl scripts were used to process raw data (raw reads) in fastq format. The next step was to remove readings containing an adapter, readings containing N bases, and readings of low quality from the raw data in order to obtain clean data (clean readings). Furthermore, Q20, Q30, and GC content clean data were calculated. All downstream analyses relied on clean data.
8
Transcriptomic Analysis of Tomato Disease Resistance
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The total RNA from disease-resistant and susceptible tomato leaves were extracted with the TIANGEN RNA kit (TIANGEN, Beijing, China). The detection of RNA integrity of samples was conducted by garose gel electrophoresis. We used a NanoPhotometer spectrophotometer (IMPLEN, Los Angeles, CA, USA) to detect the RNA sample’s purity (OD260/280 and OD260/230 ratio). Precise quantification of the RNA concentration was conducted with the Qubit2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Precise detection of the RNA integrity was conducted with the Agilent2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Using the total RNA with an amount of ≥1 μg as a template, building cDNA libraries was conducted with the Illumina NEBNext® UltraTM RNA Library Prep Kit (NEB, Ipswich, MA, USA). In total, 18 RNA-Seq libraries, namely, 6 treatments consisting of uninoculated and inoculated plants of the resistant and susceptible lines (R0, R12, R48, S0, S12, and S48) with 3 replications of each combination, were separately constructed. Initial quantification of the constructed library was performed using Qubit2.0 and the insert size of the library was tested using Agilent2100. After the test was qualified, use the Illumina platform for sequencing (San Diego, CA, USA).
9
Transcriptome Analysis of FMDV-Immunized Mouse Splenocytes
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Splenocytes from mice immunized with the FMDV antigen in saline solution or emulsified in SO-VE-GS or ISA 206 were used for transcriptome sequencing. The total RNA of the spleen was extracted with RNAprep Pure Plant Kit (Tiangen Biotech (Beijing) Co., Ltd.). The quantity and quality of RNA were measured by Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and NanoPhotometer spectrophotometer (IMPLEN, CA, USA), respectively. All technical manipulations including transcriptome sequencing and assembly were carried out by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Illumina sequencing libraries with 3 μg of RNA per sample were generated by the NEBNext UltraTM RNA Library Prep Kit (New England Biolabs, MA, USA,). Index-coded samples were clustered based on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3cBot-HS (Illumia, CA, USA). After clustering, prepared libraries were sequenced on the Illumina Hiseq platform, and then the paired-end reads with 125 bp/150 base pairs were generated.
10
Transcriptomic Analysis of M. smegmatis
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10 ml of exponentially growing cultures was harvested by centrifugation (4°C, 3000 rpm, 10 min). Total RNA was isolated from three biological replicates of each sample using a FastPrep Instrument and FastRNA Pro Blue Kits (MP Biomedicals, Solon, OH, USA) according to the manufacturer’s instructions. After treatment with DNase I, the RNA was precipitated with ammonium acetate/isopropanol. The quality and quantity of each RNA sample were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Beijing, China) and a NanoDrop 1000 spectrophotometer (Thermofisher, Shanghai, China). A Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA) was used to remove bacterial rRNA from total RNA preparations.
cDNA libraries were constructed using a NEBNextUltraTM RNA library Prep Kit (New England Biolabs, Beijing, China) and sequenced on a HiSeq 4000 sequencer (Illumina). The processed reads were mapped to the Mycobacterium smegmatis MC2 155 genome (NC_008596) using Bowtie2 v2.2.6 with default parameters. FPKM values for each gene were compared and differentially expressed genes (DEGs) were determined with HTSeq v0.6.0 and DESeq v1.22.1. Genes with a multiple hypothesis-adjusted p-value below 0.05 (adjusted p-value < 0.05) were considered as DEGs. Functional enrichment analysis was carried out using GOseq v1.22 based on the Gene Ontology database.
cDNA libraries were constructed using a NEBNextUltraTM RNA library Prep Kit (New England Biolabs, Beijing, China) and sequenced on a HiSeq 4000 sequencer (Illumina). The processed reads were mapped to the Mycobacterium smegmatis MC2 155 genome (NC_008596) using Bowtie2 v2.2.6 with default parameters. FPKM values for each gene were compared and differentially expressed genes (DEGs) were determined with HTSeq v0.6.0 and DESeq v1.22.1. Genes with a multiple hypothesis-adjusted p-value below 0.05 (adjusted p-value < 0.05) were considered as DEGs. Functional enrichment analysis was carried out using GOseq v1.22 based on the Gene Ontology database.
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