RNA sequencing was performed using a Novaseq 6000 (Illumina) sequencer according to the manual of Rhelixa Co., Ltd (Tokyo, Japan). The total RNA molecules were extracted from DLD-1 cells with or without 0.5 μM of anisomycin using an RNeasy extraction kit (QIAGEN). The total RNA samples, that showed an RNA integrity number (RIN) > 7 determined by a Bioanalyzer (Agilent), were subjected to cDNA library preparation. Then 2 μg of total RNAs were converted to cDNAs with oligo dT primers using a NEBNext Poly (A) Magnetic Isolation Module (for poly A selection, NEB) and a NEB Next Directional Ultra RNA Library Prep Kit for Illumina (for strand specific libraries, NEB). These cDNA libraries were sequenced with a Novaseq 6000 system (Illumina) to produce 150 base pair-end reads.
Nebnext poly a magnetic isolation module
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Poly(A) Magnetic Isolation Module is a product designed for the purification of polyadenylated RNA from total RNA samples. It utilizes magnetic beads coated with oligo(dT) to selectively bind and capture poly(A) RNA, allowing for its separation from other RNA species.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer, by Agilent Technologies (4 mentions)
Novaseq 6000, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nebnext multiplex oligos for index primers, by Illumina (2 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (2 mentions)
Nebnext ultra 2 rna library prep with sample purification beads, by New England Biolabs (2 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Nextseq 500 system, by Illumina (2 mentions)
Bcl2fastq v2.20 conversion software, by Illumina (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Rneasy extraction kit, by Qiagen (1 mentions)
Nebnext ultra directional rna library prep kit, by Illumina (1 mentions)
Qubit fluorometer 2, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nebnext ultra directional rna library kit, by New England Biolabs (1 mentions)
22 protocols using nebnext poly a magnetic isolation module
1
Strand-specific RNA-seq of DLD-1 cells
2
Cryptococcus neoformans RNA Sequencing
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Immediately prior to harvest, all RAW264.7 samples were imaged by light microscopy to determine the percentage of cells infected with intracellular Cn. With the exception of the mock-infected samples, Cn-infected macrophage samples were only processed further if >50% of cells were infected. Macrophages were homogenized in lysis reagent (RLT buffer, Qiagen, Germantown, MD) using a 20-gauge needle and the remaining intact Cn were removed by centrifugation at 750 x g for 5 min at 4°C. RNA was extracted from the lysates using an RNeasy® Mini Kit (Qiagen), according to the manufacturer’s instructions. Genomic DNA was then removed from the total RNA using a Message Clean kit (GenHunter, Nashville, TN) per the manufacturer’s instructions. Clean RNA was resuspended in 10 μL diethyl pyrocarbonate (DEPC)-treated water and RNA integrity and quality was appraised using a Qubit 2 fluorometer (ThermoFisher Scientific).
The cDNA libraries for RNA sequencing were prepared from 1 μg of isolated RNA using the NEBNext UltraTM Directional RNA Library Prep Kit for Illumina, the NEBNext Multiplex Oligos for Illumina Index Primers, and the NEBNext Poly(A) Magnetic Isolation Module (all from New England BioLabs, Ipswich, MA) in accordance with the manufacturer’s instructions.
The cDNA libraries for RNA sequencing were prepared from 1 μg of isolated RNA using the NEBNext UltraTM Directional RNA Library Prep Kit for Illumina, the NEBNext Multiplex Oligos for Illumina Index Primers, and the NEBNext Poly(A) Magnetic Isolation Module (all from New England BioLabs, Ipswich, MA) in accordance with the manufacturer’s instructions.
3
Comprehensive Transcriptome Analysis of C. elegans
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rRNA was removed from total RNA using Ribo-zero (Epicentre/Illumina). RNA-seq libraries were made using NEBNEXT Ultra. Single-end sequencing runs of 50 nt were done. Reads were first mapped to rRNA and tRNA using Bowtie2. Reads not mapping to rRNA and tRNA were then mapped to WBCel235 genome/WS247 annotation using Tophat2 with minimum intron size of 20 (-i), maximum intron size 50,000 (-I), and allowing for 1 mismatch (-N 1). Both guided and de novo transcriptome assembly were performed to identify new genes and transcripts. Differential expression analysis was done using Cufflinks, Cuffmerge, and Cuffdiff with multiread correct. To map to transposons and inverted repeats, featureCounts was used counting all alignments, and Deseq2 was used to assess differential expression. A maximal P value of 0.1 was used as a cutoff for differential expression.
mRNAseq was done using NEBNext Poly(A) Magnetic Isolation Module with NEBNEXT Ultra Directional RNA library kit. GO-term enrichment was analyzed using GOrilla. Statistical significance of data mined from published studies was obtained from the original publications.
mRNAseq was done using NEBNext Poly(A) Magnetic Isolation Module with NEBNEXT Ultra Directional RNA library kit. GO-term enrichment was analyzed using GOrilla. Statistical significance of data mined from published studies was obtained from the original publications.
4
Transcriptome profiling of human tissues
5
RNA Extraction from S. pombe using Phenol-Chloroform
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We extracted total RNA from S. pombe using the phenol chloroform extraction method (Castillo et al. 2003 (link)). Briefly, cells were grown to a final OD600 of 0.5. Yeast cultures (25–50 mL) were then centrifuged at 1500 rpm for 3 min and washed with H2O, and cell pellets were immediately kept on ice. Each sample was then resuspended in 0.4 mL of AE buffer (50 mM sodium acetate at pH 5.3, 10 mM EDTA at pH 8.0). Sodium dodecyl sulfate was then added to a final concentration of 1%, and proteins and DNA were extracted by adding 0.6 mL of acidic phenol/chloroform (V/V), followed by incubation for 5 min at 65°C. The aqueous phase was separated by centrifugation at 14,000 rpm for 2 min at 4°C and washed with a volume of chloroform and separated by centrifugation at 14,000 rpm for 2 min at 4°C. RNA was precipitated from the aqueous phase with ethanol. RIN quality scores were in the range of 9.6–10. We subsequently performed poly(A)+ RNA purification using the NEBNext Poly(A) magnetic isolation module and concentration with the Monarch RNA cleanup kit. The poly(A)+ purification steps were performed at the Genomics Core Facility of the Universitat Pompeu Fabra.
6
RNA Editing Assay in HEK293FT Cells
7
Illumina RNA-seq Library Preparation
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The Illumina RNA-seq library was prepared in a 96-well plate. We purified and enriched mRNA from 1 ug of total RNA using the NEBNext Poly(A) Magnetic Isolation Module (New England Biolabs, catalog no. E7490L). RNA fragmentation, first and second strand cDNA synthesis, and end-repair processing was performed using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (New England Biolabs, catalog no. E7775L). We ligated adapters in the cDNA library using adapters and unique dual indexes from the NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog no. E6440L). All procedures were performed following the manufacturer protocols. We used Qubit dsDNA BR Assay Kit (Invitrogen, catalog no. Q32853) to determine the concentration of the RNA library. The library was then pooled and qualified using the 2100 Bioanalyzer (Agilent) at Novogene, CA, USA. We sequenced the pooled library with the Illumina NovaSeq 6000 platform (150-bp paired-end reads).
8
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).
9
PolyA RNA Sequencing Protocol
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Poly A RNAs were isolated from total RNAs using NEBNext Poly(A) Magnetic Isolation Module (NEB #E7490) and then fragmented for cDNA synthesis. End repair is performed where 3′ to 5′ exonuclease activity of enzymes removes 3′ overhangs, and the polymerase activity fills in the 5′ overhangs. An ‘A’ base is then added to the 3′ end of the blunt phosphorylated DNA fragments which prepares the DNA fragments for ligation to the sequencing adapters, which have a single ‘T’ base overhang at their 3′ end. Ligated fragments are subsequently size selected through purification using the Sample Purification Beads included in the kit and undergo PCR amplification to prepare the ‘libraries. The BioAnalyzer is used for quality control of the libraries to ensure adequate concentration and appropriate fragment size free of adapter dimers. The resulting library insert size is 200bp-500bp with a median size around 300bp. Libraries were barcoded and pooled for HiSeq2500 sequencing.
10
RNA-seq Library Preparation from PBMCs
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PBMCs were isolated from fresh whole blood using Ficoll (Ficoll-Paque Plus, GE Healthcare) and total RNA was extracted from 107 PBMCs using RLT Lysis Buffer (Qiagen) by following manufacturer's instructions. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (Cat# E7765) was used to generate RNA-seq libraries. Briefly, Poly A RNAs were isolated from total RNAs using NEBNext Poly(A) Magnetic Isolation Module (NEB #E7490) and then fragmented for cDNA synthesis. End repair is performed where 3' to 5' exonuclease activity of enzymes removes 3' overhangs and the polymerase activity fills in the 5' overhangs. An “A” base is then added to the 3' end of the blunt phosphorylated DNA fragments which prepares the DNA fragments for ligation to the sequencing adapters, which have a single “T” base overhang at their 3' end. Ligated fragments are subsequently size-selected through purification using the Sample Purification Beads included in the kit and undergo PCR amplification to prepare the “libraries.” The BioAnalyzer is used for quality control of the libraries to ensure adequate concentration and appropriate fragment size free of adapter dimers. The resulting library insert size is 200–500 bp with a median size around 300 bp. Libraries were uniquely barcoded and pooled for HiSeq2500 sequencing.
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