Polyadenylated RNA was extracted from treated cells using FastTrack MAG Maxi mRNA isolation kit (Life technology, USA). RNA fragmentation Reagents (Ambion, USA) was used to randomly fragment RNA. The specific anti-m6A antibody (Synaptic Systems, 202003, 1:50) was applied for m6A pull down. The library preparation for next-generation sequencing was constructed by TruSeq Stranded mRNA Sample Prep Kit (Illumina, USA) and quantified by BioAnalyzer High Sensitivity DNA chip, and then deeply sequenced on the Illumina HiSeq 2500. For data analysis, the reads from input and m6A-IP sequencing libraries were aligned to hg19 reference genome using Tophat. Both MACS/MACS2 and exomePeak were used to call m6A peaks based on the m6A-seq bam files. To achieve high specificity, only the m6A peaks called by both MACS/MACS2 and exomePeak were retained for further analysis. Differentially methylated m6A peaks were identified according to the procedure described by Schwartz et al.51 (link). Sequence motifs enriched in m6A peak regions compared to control regions were identified using DREME.
Fasttrack magmaxi mrna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FastTrack MAGMaxi mRNA isolation kit is a lab equipment product designed for the efficient extraction and purification of mRNA from various sample types. It utilizes magnetic bead technology to capture and isolate mRNA molecules, enabling reliable and consistent mRNA recovery.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (3 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Truseq stranded mrna sample prep kit, by Illumina (1 mentions)
Bioanalyzer high sensitivity dna chip, by Illumina (1 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions)
Specific anti m6a antibody, by Synaptic Systems (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Lyticase, by Merck Group (1 mentions)
Gridion x5, by Oxford Nanopore (1 mentions)
Cloneminer 2 cdna library construction kit, by Thermo Fisher Scientific (1 mentions)
Ribo zero magnetic gold kit, by Illumina (1 mentions)
Nebnext rna library preparation kit, by New England Biolabs (1 mentions)
Bioanalyzer 2100 system, by Roche (1 mentions)
Hiseq 2000 instrument, by Illumina (1 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Rna 6000 pico kit, by Agilent Technologies (1 mentions)
15 protocols using fasttrack magmaxi mrna isolation kit
1
Profiling m6A Epitranscriptome Landscape
2
RNA Extraction from Diverse Samples
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The total RNA from CCRF-SB pellets was directly extracted with Trizol reagent (Life Technologies), according to the manufacturer's protocol. For yeast and E. coli, lysis was performed with lyticase (Sigma) and lysozyme (Fluka), respectively, before total RNA was extracted with Trizol reagent as described earlier. Dengue virus genomic RNA was extracted from purified viral particles using Trizol reagent (42 (link)). The poly(A)-tailed RNA in human and yeast cells was isolated from the total RNA using a Fasttrack MAG Maxi mRNA isolation kit (Life Technologies) following the manufacturer's protocol. All RNA samples were used immediately or stored at −80°C.
3
RNA Extraction from M. foliosa
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All the RNA extraction procedures followed the manufacturer’s instructions. The total RNA was isolated with TRIzol LS Reagent (Thermo Fisher Scientific, 10296028, Waltham, MA, USA) and treated with DNase I (Thermo Fisher Scientific, 18068015). The high-quality mRNA was isolated with a FastTrack MAG Maxi mRNA Isolation Kit (Thermo Fisher Scientific, K1580-02). The samples were separated from healthy M. foliosa to ensure that enough high-quality RNA (>10 µg) could be obtained for a full-length cDNA transcriptome library. To support the accuracy and credibility of the data, we used three sample repetitions for the library construction and sequencing. Each sample was collected from an independent colony.
4
Thawing and Sequencing of Frozen Blood Cells
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Viably frozen diagnostic peripheral blood cells were thawed at 37 °C followed by suspension in phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum. After the cells were washed, mRNA was extracted using the Fast track MAG – maxi mRNA isolation kit (Thermo Fisher Scientific) and prepared for sequencing on the GridION X5 (Oxford Nanopore Technologies) according to the manufacturer’s protocol. The sequencing data was then mapped to the human reference genome (hg38) using Minimap2 (v2.17-r941), after which it was inspected with the IGV (v2.8.2) software51 .
5
RNA Extraction and cDNA Synthesis Protocol
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The total RNA of A. cinnamomea was extracted by TRIzol method [8 ], and the total RNA integrity and quality analysis detected by
electrophoresis on a 1% agarose gel and Nanodrop2000C (Thermo, USA). The mRNA was isolated by
the FastTrack MAG Maxi mRNA Isolation Kit (Thermo, USA). cDNA synthesis was performed by the
CloneMiner II cDNA Library Construction Kit (Thermo, USA).
electrophoresis on a 1% agarose gel and Nanodrop2000C (Thermo, USA). The mRNA was isolated by
the FastTrack MAG Maxi mRNA Isolation Kit (Thermo, USA). cDNA synthesis was performed by the
CloneMiner II cDNA Library Construction Kit (Thermo, USA).
6
Transcriptome Analysis of m6A Methylation
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Total RNA was isolated from HOS cells using the TRIzol reagent (Thermo Fisher Scientific) and FastTrack MAGMaxi mRNA isolation kit (Thermo Fisher Scientific). RNA fragmentation, m6A-seq and library preparation were commissioned to CloudSeq Biotech (Shanghai, China). Library preparation was performed using the NEBNext Super Directional RNA Library Preparation Kit (New England BioLabs, Ipswich, MA, USA). Significant peaks with false discovery rate (FDR) <0.01 were obtained and annotated into the RefSeq database, and sequences were identified using Homer (Hypergeometric Optimization of Motif EnRichment). Cuffdiff (Cufflinks, USA) was used to find the corresponding modified genes.
HOS cells with low expression of METTL3 and control cells were pre-hybridized with DNA. Whole transcriptome libraries were prepared using the Ribo-Zero Magnetic Gold kit (Illumina, San Diego, CA, USA) and the NEBNext RNA Library Preparation Kit (New England Biolabs). Quality control and quantification were performed by BioAnalyzer 2100 system (Kapa Biosystems, USA), and the resulting libraries were sequenced and analyzed for differentially expressed mRNAs on a HiSeq2000 instrument (Illumina).
HOS cells with low expression of METTL3 and control cells were pre-hybridized with DNA. Whole transcriptome libraries were prepared using the Ribo-Zero Magnetic Gold kit (Illumina, San Diego, CA, USA) and the NEBNext RNA Library Preparation Kit (New England Biolabs). Quality control and quantification were performed by BioAnalyzer 2100 system (Kapa Biosystems, USA), and the resulting libraries were sequenced and analyzed for differentially expressed mRNAs on a HiSeq2000 instrument (Illumina).
7
m6A Profiling of MKN-45 Cell Line
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Total polyadenylated RNA was isolated from MKN-45 cells by using FastTrack MAGMaxi mRNA isolation kit (Thermo). RNA fragmentation, m6A RIP, and library preparation were conducted according to manufacturer’s instructions and previously published protocol [31 (link)]. The library was prepared by use of NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs). Each experiment was conducted with two biological replicates. m6A-seq data were analyzed according to protocols described before [31 (link)]. Significant peaks with FDR < 0.05 were annotated to RefSeq database (Hg19). Sequence motifs were identified by using Homer. Gene expression was calculated by Cufflinks using sequencing reads from input samples. Cuffdiff was used to find DE genes.
8
RNA Extraction for Coral Transcriptomes
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All RNA extraction procedures were carried out according to the manufacturer’s instructions. Total RNA was isolated with TRIzol LS Reagent (Thermo Fisher Scientific, 10296028) and treated with DNase I (Thermo Fisher Scientific, 18068015). High-quality mRNA was isolated with a FastTrack MAG Maxi mRNA Isolation Kit (Thermo Fisher Scientific, K1580-02). Samples were separated from healthy P. damicornis, P. verrucosa, M. capricomis, and A. muricata to ensure that enough high-quality RNA (>10 μg) could be obtained for a full-length cDNA transcriptome library.
9
PolyA+ RNA Extraction from Worms
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PolyA+ RNA was extracted from eggs, from 200 adult male worms and from 200 adult female worms using two rounds of the FastTrack MAG Maxi mRNA Isolation Kit (Invitrogen, Thermo Scientific, USA), as described [6 (link)], with the following modifications: 1,400 units of RNase Out (Invitrogen) and 20 mM of Vanadyl Ribonucleoside Complexes (VRC) were added to Lysis Buffer L4 at the first step of the sample preparation; at the final binding step, an additional 1,400 units of RNase Out (Invitrogen) was added to the sample while the tube remained in the rotator; treatment with 30 units of DNase I Amplification Grade (Invitrogen) was performed for 45 min at room temperature; and six washings were performed at the final washing step before elution. These modifications resulted in PolyA+ RNA samples with small percentages of rRNA contamination (3 to 7%), as estimated with a Bioanalyzer using the RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).
10
m6A-Seq for Transcriptome-Wide Mapping of N6-Methyladenosine
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The total polyadenylated RNA was isolated from HeLa cells treated with or without 10 ng/ml TGF-β for 3 days using TRIZOL reagent followed by isolation through FastTrack MAGMaxi mRNA isolation kit (Invitrogen). RNA fragmentation, m6A-seq, and library preparation were performed according to the manufacturer’s instructions and previously published protocol7 (link). NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) was used for library preparation. Each experiment was conducted with two biological replicates. m6A-seq data were analyzed according to protocols described before7 (link). Significant peaks with FDR < 0.05 were annotated to RefSeq database (hg19). Sequence motifs were identified by using Homer. Gene expression was calculated by Cufflinks using sequencing reads from input samples. Cuffdiff was used to find DE genes.
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