RNA was extracted using Direct-zol RNA extraction kit (Zymo Research) from two biological replicates for each sample of cells at PPs and pancreatic islets stages of differentiation. mRNA was captured from 1 μg of total RNA using NEBNext (Poly A) mRNA magnetic isolation kit (NEB, E7490) according to the manufacturer’s instructions. NEBNext ultra directional RNA library prep kit (NEB, E7420L) used to NEBNext ultra directional RNA library prep kit (NEB, E7420L) used to prepare RNA-seq libraries which is sequenced on an Illumina Hiseq 4000 system. The initial processing of the raw data involved basic trimming and quality control, which was carried out using Illumina BCL2Fastq Conversion Software v2.20.
Nebnext poly a mrna magnetic isolation kit
Manufactured by New England Biolabs
Sourced in United Kingdom
The NEBNext Poly(A) mRNA Magnetic Isolation Kit is a lab equipment product designed to isolate polyadenylated (poly(A)) mRNA from total RNA samples using magnetic beads. The kit provides the necessary reagents and protocol for efficient purification of poly(A) mRNA from a variety of sample types.
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Lab products found in correlation
Hiseq 2500, by Illumina (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (2 mentions)
Hiseq 4000 system, by Illumina (2 mentions)
Bcl2fastq conversion software v2, by Illumina (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (2 mentions)
Rabbit anti m6a antibody, by Synaptic Systems (2 mentions)
Vahts library quantification kit for low rox premixed, by Illumina (2 mentions)
Nebnext ultra directional rna library prep kit for, by Illumina (2 mentions)
Direct zol rna extraction kit, by Zymo Research (1 mentions)
Hiseq 2500 machine, by Illumina (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Nebnext rna library prep kit, by New England Biolabs (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Kapa stranded rna seq library preparation kit, by Roche (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Quant it dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nebnext rna library prep kit for, by Illumina (1 mentions)
16 protocols using nebnext poly a mrna magnetic isolation kit
1
RNA-seq library preparation protocol
2
Mapping of m6A Epitranscriptomic Landscape
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MeRIP-sequencing projects and subsequent data analysis were supported by Genesky Biotechnologies Inc. (Shanghai, China). Total RNA was isolated from SW480 cells transfected with shMETTL14-2 (shM14-2) or shControl (shCON), followed by poly (A) + RNA purification and fragmentation using NEBNext Poly (A) mRNA Magnetic Isolation Kit (New England Biolabs, UK). Concentration of RNA was detected on Nanodrop 2000 (Thermo Fisher Scientific, USA) and the integrality was guaranteed by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Dynabeads Protein A (Thermo Fisher Scientific, USA) was mixed with rabbit anti-m6A antibody (Synaptic system, Germany) at 4 °C for 2 h in advance, then fragmented mRNA was incubated with the mixture for another 2 h to precipitate m6A-enriched RNAs. Qualified samples underwent Library Pooling and Sequencing using Illumina HiSeq 2500 machines. Following quality filter, the raw sequence data was mapped to human genome GRCh37/hg19 utilizing the HISAT2 software (v2.0.5) and the results were subjected to analyzed bioinformatically and statistically. The peak calling data and RNA-sequencing data were described in Supplementary Materials .
3
RNA Extraction and Sequencing of Motor Neurons
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Total RNA was extracted from day 6 of differentiated motor neurons using TRIzol reagent (Invitrogen, 15596026). NEBNext Poly(A) mRNA Magnetic Isolation Kit (NEB, E7490) was used to remove ribosomal RNA followed by DNase treatment. Total RNA for primary cortical neurons was extracted after 6 days in vitro culture using TRIzol reagent. RNA-seq libraries were prepared for sequencing using NEBNext RNA Library Prep Kit (NEB, E7765) and were sequenced by Illumina HiSeq 2500 and NovaSeq 6000 System.
4
Infant Transcriptome Sequencing Protocol
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PolyA-enrichment of total RNA was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Kit (New England Biolabs), followed by cDNA library synthesis using the KAPA Stranded RNA-seq Library Preparation Kit as per the manufacturer’s instructions (Kapa Biosystems). Quantification of the cDNA libraries was performed using a Quant-iT dsDNA Assay Kit (Invitrogen) and normalized to 4 nM. To derive the transcriptome for each infant, samples were labelled with a unique barcode, multiplexed, and sequenced on a HiSeq 2500 sequencer (Illumina), using the High Output 100-bp single-end run, at the University of British Columbia Sequencing Centre.
The deconvoluted sequences from each infant were analysed and the quality of FASTQ reads were assessed using FastQC(v0.0.15), and summarized using MultiQC(v0.8.dev0) [20 , 21 (link)]. FASTQ reads were aligned to the GRCh37(hg19) human reference genome [22 (link)] using the STAR(v2.5) aligner, followed by generation of read counts using htseq-count(v0.6.1p1) [23 (link)]. Globin transcripts and low count genes were bioinformatically removed from the count matrix. The median library size of uniquely mapped reads for very preterm infant samples was 6.8 million reads, and a range of 4.1–8.9 million reads for the entire study.
The deconvoluted sequences from each infant were analysed and the quality of FASTQ reads were assessed using FastQC(v0.0.15), and summarized using MultiQC(v0.8.dev0) [20 , 21 (link)]. FASTQ reads were aligned to the GRCh37(hg19) human reference genome [22 (link)] using the STAR(v2.5) aligner, followed by generation of read counts using htseq-count(v0.6.1p1) [23 (link)]. Globin transcripts and low count genes were bioinformatically removed from the count matrix. The median library size of uniquely mapped reads for very preterm infant samples was 6.8 million reads, and a range of 4.1–8.9 million reads for the entire study.
5
RNA-seq transcriptome profiling pipeline
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Total RNA was extracted using TRIzol (Invitrogen), and mRNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation kit (New England Biolabs). Reverse transcription was carried out, and cDNA was fragmented, purified, and ligated with adapters. PCR was performed using an End Repair/dA-tail ligated DNA template to include unique barcodes to identify each sample. The samples were sequenced as 125-bp pair-end reads using a HiSeq 2500 instrument (Illumina) with two biological replicates. The reads were mapped to the hg19 reference genome using TopHat2 (Kim et al. 2013 (link)). Next, Cufflinks was used to estimate gene expression (FPKM) with alignments (Trapnell et al. 2010 (link)). Finally, DESeq2 was used to estimate the differential genes between two cell conditions with the count number of each gene (Love et al. 2014 (link)).
6
RNA-Seq Library Preparation and Sequencing
7
RNA-seq Library Preparation from Mouse Samples
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We fed 1 μg total RNA from mice sample into the NEBNext PolyA mRNA Magnetic Isolation Kit (NEB, catalog #E7490L), then constructed the specific chain RNA library using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, catalog #E7420L). We performed library construction according to the vendor’s instructions, starting with the chapter “Protocol for use with NEBNext Poly (A) mRNA Magnetic Isolation Module.” The mRNA is enriched by magnetic beads, followed by first- and second-strand cDNA synthesis. Double-stranded cDNA was purified using Agencourt AMPure XP Beads for cDNA library construction. The quality of the library was evaluated on the Agilent 2100 bioanalyzer and quantified by qPCR using the VAHTS library quantification Kit for Illumina (Low ROX Premixed) (Vazyme, catalog #NQ103). Libraries were sequenced on the HiSeq X10 using the paired end 2*150bp, single-index format. The RNA-seq data were deposited in the National Center for Biotechnology Information (NCBI) under accession code PRJNA759363 and PRJNA835059.
8
RNA-seq Library Preparation and Sequencing
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Total RNA was isolated from cells using Trizol (Sigma-Aldrich). We fed 1 μg RNA into the NEBNext PolyA mRNA Magnetic Isolation Kit (New England Biolabs, catalog number E7490L) and then constructed the specific chain RNA library using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, catalog number E7420L). We performed library construction according to the vendor’s instructions, starting with the chapter “Protocol for use with NEBNext Poly (A) mRNA Magnetic Isolation Module.” Library quality was evaluated on an Agilent 2100 bioanalyzer and quantified by qPCR using the VAHTS Library Quantification Kit for Illumina (Low ROX Premixed) (Vazyme, catalog number NQ103). Libraries were sequenced on the HiSeq ×10 using the paired-end 2 × 150 bp, single-index format. The RNA-seq data have been deposited in the National Center for Biotechnology Information (NCBI) under accession code PRJNA759363 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA759363/ ).
9
MeRIP-seq Analysis of METTL16 Knockdown
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Total RNA was extracted from SW620 cells transfected with shMETTL16‐2 or shNC using TRIZOL reagent, followed by poly (A) + RNA purification and fragmentation using the NEBNext poly (A) mRNA Magnetic Isolation Kit (New England Biolabs, UK). MeRIP‐sequencing and data analysis were supported by Genesky Biotechnologies Inc (Shanghai, China). Qualified samples underwent Library Pooling and Sequencing using Illumina HiSeq 2500 machines. Following quality filter, the raw sequence data were mapped to human genome GRCh37/hg19 utilizing the HISAT2 software (v2.0.5) and the results were subjected to analysed bioinformatically and statistically.
10
Profiling YTHDF2 KO RNA-seq and m6A MeRIP-seq
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For RNA-seq, total RNA from SW780 cells with stable expression of YTHDF2 KO and corresponding control cells were extracted using TRizol Reagent and submitted to the Beijing Genome Institute (BGI) for library construction and sequencing. Total RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer and NanoDrop 2000. mRNAs were enriched using oligo(dT)-attached magnetic beads and subsequently fragmented into small pieces. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The mRNA libraries were then sequenced on a BGISEQ-500 platform.
Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was conducted by LC-Bio. Briefly, mRNA was purified using the NEBNext Poly(A) mRNA Magnetic Isolation Kit (E7490S, NEB). RNA fragmentation was performed by incubation with magnesium ions at 94°C using NEBNext Magnesium RNA Fragmentation Module (E6150S, NEB). The specific anti-m6A antibody was applied for m6A pull down. The enriched mRNA fragments were then used to construct libraries with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (NR603, Vazyme). Sequencing was carried out on an Illumina HiSeq 2500 with paired-end 150-bp read length.
Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was conducted by LC-Bio. Briefly, mRNA was purified using the NEBNext Poly(A) mRNA Magnetic Isolation Kit (E7490S, NEB). RNA fragmentation was performed by incubation with magnesium ions at 94°C using NEBNext Magnesium RNA Fragmentation Module (E6150S, NEB). The specific anti-m6A antibody was applied for m6A pull down. The enriched mRNA fragments were then used to construct libraries with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (NR603, Vazyme). Sequencing was carried out on an Illumina HiSeq 2500 with paired-end 150-bp read length.
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