RNA was isolated using the Ambion® RNaqueous® micro kit (ThermoFisher Scientific) according to manufacturer’s instructions. RNA quality was analyzed using the Agilent 6000 Pico LabChip® and Agilent 2100 Bioanalyzer software (Agilent Technologies, USA). RNA quantity was determined using the Quant-iT™ RiboGreen® kit (ThermoFisher Scientific) according to manufacturer’s instructions. NEBNext Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, USA) were used for mRNA purification. cDNA library synthesis was performed according to Ziegler et al.64 (link) with three biological replicates per treatment each containing three A. thaliana leaves. Subsequently, 100 bp paired-end RNA sequencing was performed on the Illumina HiSeq4000 platform (Génome Québec Innovation Centre, McGill University, Montreal, Canada).
Nebnext oligo dt 25 beads
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Oligo(dT)25 beads are magnetic beads coated with oligo(dT)25 sequences. They are designed for the isolation and purification of mRNA from total RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000 platform, by Illumina (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq x ten, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Qubit 2, by Thermo Fisher Scientific (3 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Nebnext rna first and second strand synthesis module, by New England Biolabs (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Quant it ribogreen kit, by Thermo Fisher Scientific (1 mentions)
Agilent 6000 pico labchip, by Agilent Technologies (1 mentions)
Ambion rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
Qubit dna hs assay, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nebnext ultra 2 rna library prep kit, by New England Biolabs (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
10 protocols using nebnext oligo dt 25 beads
1
Transcriptomic Analysis of Arabidopsis Leaves
2
RNA-seq Analysis of Plant Roots
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The root sample collected above from one root per cultivar or landrace was used for transcriptome sequencing. Zhang et al. (2019) (link) showed that the expressions of gene transcripts quantified by RNA-seq were highly reproducible, varying from correlation coefficient r = 0.90 to 0.98 (P < 0.0001) between plants collected from a field trial replicate, different field trial replicates, and across years. Therefore, the mRNA of one plant root was sequenced for this study. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany), following the manufacturer’s protocol. The integrity and quantity of the RNAs were estimated by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Poly(A) mRNA was purified from total RNA by NEBNext Oligo(dT)25 beads (NEB, USA). cDNA libraries were constructed from the mRNAs using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), as described by the manufacturer. Library sequencing was accomplished using the Illumina HiSeq 4000 platform with a 150-base paired-end (150PE) module at Gene Denovo Biotechnology Co (Beijing, China). Raw reads of low quality were removed, including those with adapters, containing more than 10% unknown nucleotides, and containing more than 50% low-quality bases (
3
RNA Isolation and Sequencing
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Libraries were prepared using RNA of cells treated either with the vehicle or with Sfb. Total RNA was prepared as described above. Then, concentration and quality of the RNA were assessed with Qubit (Qubit™ DNA HS assay, Thermo Fisher Scientific, Waltham, MA, USA) and a 2100 Bioanalyzed Nano Chip (Agilent Technologies Genomics, Santa Clara, CA, USA), respectively. RNA Integrity Number (RIN) values were > 9 in all RNA samples. Polyadenylated RNA was isolated from the total RNA using NEBNext Oligo d(T)25 beads (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Samples were normalized to an equivalent concentration of 67.3 ng/µL and prepared for RNA Ilumina Sequencing. For HepG2 cell lines, three biological replicates were obtained, while for SNU423 cells only two biological replicates were collected.
4
Illumina Sequencing of Total RNA
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Total RNA was prepared with QIAzol Lysis Reagent and RNeasy MinElute Cleanup Kit (Qiagen, Germany). The RNA quality and integrity were analyzed by Qubit 2.0(Life Technologies, USA) and Bioanalyzer 2100 (Agilent, Germany). For library preparation, 3 μg total RNA were captured by NEBNext Oligo d(T) 25 beads (NEB, USA), sheared to fragments of ~ 250 bp, and reverse transcripted by NEBNext RNA first and second Strand Synthesis Module (NEB, USA). The products were end-repaired, A-tailed, and ligated to Illumina sequencing adapters and amplified by PCR. The sequencing library were qualified by Qubit 2.0 (Life technologies, USA) and Bioanalyzer 2100 (Agilent, Germany), then sequenced on Illumina Hiseq X-Ten with 2 × 150 bp paired-end sequencing, which were controlled by Hiseq Control Software (HCS).
5
RNA-seq Library Preparation and Analysis
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The RNA quality and integrity were analyzed by Qubit 2.0 (Life Technologies) and Bioanalyzer 2100 (Agilent). For library preparation, 3 μg total RNA was captured by NEBNext Oligo d (T) 25 beads (NEB), sheared to yield fragments of approximately 250 bp, and reverse transcribed using NEBNext RNA first and second Strand Synthesis Module (NEB, USA). The products were end-repaired, A-tailed, ligated to Illumina sequencing adapters and amplified by PCR. The quality of the sequencing library was assayed by using the Qubit 2.0 fluorometer (Life Technologies, USA) and the Bioanalyzer 2100 (Agilent) and then sequenced using an Illumina Hiseq X Ten with 2 × 150 bp paired-end sequencing, controlled by HiSeq Control Software (HCS). Raw sequence reads were initially examined using FastQC for quality control. Raw reads were processed to trim low-quality sequences and adapters using Trimmomatic. Clean reads were then mapped to hg19 for human samples and mm9 for mouse samples using STAR, and only uniquely mapped reads were kept. Read counts were calculated by htseq-count. Differential expression analysis was performed using DESeq2.
6
Transcriptome Analysis of Medicago sativa
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Total RNA was extracted from three biological replicates of the leaves of each M. sativa variety using the RNeasy Plant Mini Kit (Qiagen, Germany), following the manufacturer’s protocol. The integrity of RNA samples was measured by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The Poly (A) mRNA was enriched by NEBNext Oligo(dT)25 beads (NEB, USA) from 50 μl total RNA. Then, the enriched mRNA was constructed to a cDNA library by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), following the manufacturer’s protocol.
7
Illumina RNA-seq Library Preparation and Analysis
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The RNA quality and integrity were analyzed by Qubit 2.0 (Life Technologies) and Bioanalyzer 2100 (Agilent). For library preparation, 3 μg total RNA was captured by NEBNext Oligo d (T) 25 beads (NEB), sheared to yield fragments of approximately 250 bp, and reverse transcribed using NEBNext RNA first and second Strand Synthesis Module (NEB, USA). The products were end-repaired, A-tailed, ligated to Illumina sequencing adapters, and amplified by PCR. The quality of the sequencing library was assayed by using the Qubit 2.0 fluorometer (Life technologies, USA) and the Bioanalyzer 2100 (Agilent) and then sequenced using an Illumina Hiseq X Ten with 2 × 150 bp paired-end sequencing, controlled by HiSeq Control Software (HCS). Raw sequence reads were initially examined using FastQC for quality control. Raw reads were processed to trim low-quality sequences and adapters using Trimmomatic. Clean reads were then mapped to hg19 for human samples and mm9 for mouse samples using STAR, and only uniquely mapped reads were kept. Read counts were calculated by htseq-count. Differential expression analysis was performed using DESeq2.
8
RNA-seq Analysis of PRDM1 Overexpression
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GFP+ cells from LV-PRDM1 groups or LV-Control groups were isolated by flow cytometric sorting. RNA was extracted using the RNeasy Micro Kit (QIAGEN, 74004) and purified by NEBNext Oligo d(T)25 beads (NEB, E7490). NEBNext Ultra II RNA Library Prep Kit (NEB, E7770S) was used to generate cDNA libraries. AMPure XP beads (Beckman Coulter, A63881) were used to purified cDNA libraries between 300 and 500bp. Paired-end sequencing was performed on a NovaSeq6000 (Illumina), producing between 28 and 35 million 150-bp pair-end reads per sample.
The quality of the raw fastq files was examined with the FastQC software. Raw fastq files were trimmed using the software Trimmomatic by setting the parameter “LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4:15 MINLEN: 36”. The trimmed fastq files were then aligned to the human GRC38/hg38 reference genome using Hisat2, and gene expression was quantified by StringTie (18 (link)). Then differential expression analysis was run using DESeq2 package. GO enrichment analysis was performed using DAVID (https://david.ncifcrf.gov/ ). GSEA was performed using the Broad Institute software (http://software.broadinstitute.org/gsea/index.jsp ). Enrichment scores were calculated by comparing LV-PRDM1 to LV-Control groups.
The quality of the raw fastq files was examined with the FastQC software. Raw fastq files were trimmed using the software Trimmomatic by setting the parameter “LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4:15 MINLEN: 36”. The trimmed fastq files were then aligned to the human GRC38/hg38 reference genome using Hisat2, and gene expression was quantified by StringTie (18 (link)). Then differential expression analysis was run using DESeq2 package. GO enrichment analysis was performed using DAVID (
9
RNA-seq Transcriptome Assembly Protocol
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Roughly 100 mg of frozen tissue was then used for total RNA extraction with an OmniPlant RNA Kit based upon provided directions. For RNA-seq analyses, NEBNext Oligo(dT)25 beads (NEB, USA) were used to speci cally enrich for the mRNA present within a 50 μl total RNA sample, after which a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) was used to prepare an mRNA library from this enriched samples according to provided directions. An Illumina HiSeq TM 4000 platform was then used for sequencing. The resultant raw reads then underwent quality ltering in order to remove those reads that were of low-quality, contained poly-N sequences, or contained adapter sequences. Trinity was then used for de novo assembly of clean reads [53] , yielding a transcriptomic reference database.
10
Transcriptome Profiling of Plant Tissues
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Roughly 100 mg of frozen tissue was then used for total RNA extraction with an OmniPlant RNA Kit based upon provided directions. For RNA-seq analyses, NEBNext Oligo(dT)25 beads (NEB, USA) were used to specifically enrich for the mRNA present within a 50 μl total RNA sample, after which a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) was used to prepare an mRNA library from this enriched samples according to provided directions. An Illumina HiSeq TM 4000 platform was then used for sequencing. The resultant raw reads then underwent quality filtering in order to remove those reads that were of low-quality, contained poly-N sequences, or contained adapter sequences. Clean reads were then de novo assembled with Trinity [51] (link), thereby producing a transcriptomic reference database.
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