Three replicate total RNAseq libraries from 5 ng HBRR input each were prepared using the NEBNext Ultra RNA Library Kit for Illumina (NEB) with omission of the mRNA isolation step. Instead, fragmentation was performed directly on total RNA by adding 4 μl of first strand reaction buffer (5×) and 1 μl random primers to 5 μl of the 1 ng/μl HBRR/ERCC mix (see above) and heating the mixture at 94°C for 15 min. Afterward, the manufacturer's instructions were followed and final libraries were amplified with 13 cycles.
Nebnext ultra rna library kit
Manufactured by Illumina
Sourced in United States
The NEBNext Ultra RNA Library Kit is a laboratory equipment product designed for preparing RNA samples for next-generation sequencing (NGS) applications. It provides a streamlined workflow for generating high-quality cDNA libraries from a variety of input RNA sources.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 device, by Illumina (1 mentions)
Tophat, by Illumina (1 mentions)
Nebnext rrna depletion kit, by New England Biolabs (1 mentions)
Rneasy mini column, by Qiagen (1 mentions)
Hiseq 3000 sequencer, by Illumina (1 mentions)
Agilent 2000 bioanalyzer, by Agilent Technologies (1 mentions)
Mirvana kit, by Thermo Fisher Scientific (1 mentions)
Nebnext poly a magnetic isolation module, by New England Biolabs (1 mentions)
Rna 6000 nano chip, by Agilent Technologies (1 mentions)
Polya spin mrna isolation kit, by New England Biolabs (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Na ve t cell isolation kit, by Miltenyi Biotec (1 mentions)
9 protocols using nebnext ultra rna library kit
1
Total RNA-seq from Nanogram Input
2
RNA-seq analysis of tumor-infiltrating CD8+ T cells
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Tumor-infiltrating CD8 T cells from CT26-tumor bearing mice treated with Glucose 5% (control) or Folfox when tumors reached 50–70 mm2 were sorted by flow cytometry 8 days following treatment. CD8 TILs cells coming from 2 independent experiments with 8–10 pooled tumors for each experiment were used. Splenic CD8 T cells from 2 naïve mice were used as reference. Total mRNA was isolated using Trizol (Gibco Life Technologies) according to the manufacturer's instructions. rRNA from total RNA extracted were removed with NEBNextrRNA depletion kit (New England BioLabs). 100 ng of RNA depleted of rRNA was used for the library preparation with a NEBNext Ultra RNA library kit for Illumina according to the manufacturer's instructions (New England BioLabs). RNA sequencing was performed on a NextSeq 500 device (Illumina). The libraries were sequenced with paired-end 75–base pair ‘reads’. FASTQ files were mapped with the BWA software package (mm10 National Center for Biotechnology Information assembly of the Mus musculus genome) for Illumina. Analysis was performed with the splice junction mapper TopHat for Illumina. The files generated were processed with Cufflinks software to obtain annotated expressed genes in each studied subtype. Heatmaps of selected genes were generated using R software (http://www.R-project.org ).
3
Wt1 Knockout RNA Sequencing
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RNA from mouse ES cell line E14, Wt1 knockout ES line (KO1A), M15 control, a stable lentiviral Wt1 knockdown M15 cell line, and a control lacZ lentiviral stable line was isolated using the Qiagen RNeasy minicolumns as per the manufacturer's protocol. The isolated total RNA (1 µg) was poly A-selected and subjected to library preparation with the NEBnext Ultra RNA library kit for Illumina sequencing.
4
RNA Extraction and Sequencing Workflow
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The WBC RNA was extracted using the mirVanaTM kit (Ambion, Carlsbad, CA). The quality of the mRNA was first checked using a Nanodrop (Thermo Fisher Scientific, Willington, DE). If samples had a measured λ260/λ280 > 1.9, they were run on Agilent 2000 bioanalyzer using the RNA 6000 Nano chip (Agilent, Santa Clara, CA). Samples with RIN # > 7.0 with 1–10 μg of total RNA were used to prepare libraries.
The libraries were constructed using the NEB Next Ultra RNA library kit for Illumina with the NEBNext PolyA Magnetic Isolation Module (NEB, Ipswich, MA). After final clean-up, 1 μl of the libraries were run on an Agilent 2000 bioanalyzer using the high sensitivity DNA chip. Based on the average size and concentration, the individual libraries were pooled to an equal molar concentration. The pools were sequenced using an Illumina HiSeq 3000 sequencer and were run at 2× 100 bp at the Iowa State University DNA Sequencing Facility (Ames, IA, USA).
The libraries were constructed using the NEB Next Ultra RNA library kit for Illumina with the NEBNext PolyA Magnetic Isolation Module (NEB, Ipswich, MA). After final clean-up, 1 μl of the libraries were run on an Agilent 2000 bioanalyzer using the high sensitivity DNA chip. Based on the average size and concentration, the individual libraries were pooled to an equal molar concentration. The pools were sequenced using an Illumina HiSeq 3000 sequencer and were run at 2× 100 bp at the Iowa State University DNA Sequencing Facility (Ames, IA, USA).
5
Renilla Transcriptome Profiling and Assembly
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Renilla total RNA was prepared from 2 live animals by freezing in liquid nitrogen, homogenization by mortar and pestle, and Trizol extraction. Poly-A+ RNA was isolated from 600 μg total RNA using the poly-A Spin mRNA isolation kit from New England Biolabs (NEB) using two consecutive oligo-dT selections following the kit protocols. 50 ng of poly-A+ RNA was used to construct a directional cDNA library using the NEBNext Ultra RNA library kit for Illumina following the kit instructions. The fragmentation step was performed for 13 min at 94°C. Final amplification was performed for 15 cycles and the library was purified from primers and adaptors using two consecutive Ampure bead steps before quality assessment in an Agilent Bioanalyzer which indicated a library with a peak corresponding to 262 bp.
Sequencing of the library was performed in the Illumina MiSeq. 12 million reads were obtained.
The reads were used as input for the Trinity assembly software [12 (link)] for de novo transcript assembly, resulting in 48,880 assembled RNA transcripts. The corresponding DNA sequences were generated. The entire assembled cDNA library generated from Trinity was translated in all 3 frames.
Sequencing of the library was performed in the Illumina MiSeq. 12 million reads were obtained.
The reads were used as input for the Trinity assembly software [12 (link)] for de novo transcript assembly, resulting in 48,880 assembled RNA transcripts. The corresponding DNA sequences were generated. The entire assembled cDNA library generated from Trinity was translated in all 3 frames.
6
Microglia RNA-Seq: Revealing IgG-Induced Transcriptional Changes
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RNA was extracted from microglia treated with PBS or IgG (10 µg/ml) at DIV15 for 24 h. After QC, mRNA was enriched using oligo(dT) beads, and rRNA was removed using the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA). First, the enriched mRNA was randomly fragmented using fragmentation buffer, followed by cDNA synthesis using the NEB Next Ultra RNA Library Kit (Illumina), according to the manufacturer’s protocols. The qualified libraries were pooled and fed into NovaSeq6000 sequencers (Illumina) according to the recommended pooling protocol based on the effective concentration and anticipated data volume. The filtering process for cleaning reads included the removal of reads containing adapters, those with N > 10% (where N represents an undetermined base), and those with low-quality bases (Q score ≤ 5), accounting for > 50% of the total bases. Clean reads were aligned with the mouse reference genome mm10 using hisat2 [28 (link)]. The p-value was calculated using the population stability index distribution for each group as the input. The p-value for this event was < 0.0001, considering the clear separation of the two distributions. In total, 373 DEGs (261 upregulated and 112 downregulated genes) with p-values < 0.01 and |logFC| > 0.5 were further analyzed using the Qiagen Ingenuity pathway analysis (IPA) software (version 81,348,237; Qiagen, Hilden, Germany).
7
RNA-seq Analysis of Th1 and Th17 Lymphocytes
8
RNA-Seq Library Preparation and Quantification
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The 19 RNA-Sequencing directional libraries were prepared according to the NEBNext Ultra RNA library kit for Illumina sequencing, using poly-A mRNA magnetic isolation (New England Biolabs, MA, USA). The sequencing process was carried out in one single lane of an Illumina HiSeq 4000 platform, generating 150 base-paired end reads.
The quality of raw and cleaned sequences was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality filtering and adapter removal were performed using a Trimmomatic v.0.35 [83 (link)]. The reads were aligned using STAR v.2.4.0.1 [84 (link)] to the chicken Ensembl reference genome (Gallus gallusv.5.0).
ReadCounter (http://www.genefriends.org/ReadCounter/references/) was used to quantify the number of read mappings on each gene locus using Galgal5 Ensembl (90) annotation coordinates.
The quality of raw and cleaned sequences was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality filtering and adapter removal were performed using a Trimmomatic v.0.35 [83 (link)]. The reads were aligned using STAR v.2.4.0.1 [84 (link)] to the chicken Ensembl reference genome (Gallus gallusv.5.0).
ReadCounter (http://www.genefriends.org/ReadCounter/references/) was used to quantify the number of read mappings on each gene locus using Galgal5 Ensembl (90) annotation coordinates.
9
Transcriptomic Profiling of Naive CD4+ T Cells
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Total naïve CD4+ T cells were isolated from the spleen of WT and Pcbp1f/f Cd4-Cre mice using the naïve T cell isolation kit (Miltenyi, USA). Total RNA was extracted from the cells and sent to Novogene for RNA-seq analysis. Libraries were prepared using 50 ng of total RNA using the NEBNext Ultra RNA Library Kit for Illumina (catalog no. E7530) and sequenced on HiSeq 3000 at 75 base pair paired-end. Each sample was analyzed in quadruplet. Sequencing reads were first evaluated for quality control using the FastQC software and then aligned against the mouse mm10 reference genome using the TopHat2 software. Mapped reads were assigned to gene features and quantified using the HTSeq software and further evaluated for quality control using correlation and multidimensional scaling plots. Normalization and differential expression were performed using the edgeR software. The significantly DEGs (FDR < 0.05) were visualized using heat maps and volcano plot representations and considered for subsequent functional enrichment analysis using ToppGene Suite.
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