High integrity (>8.5 Agilent Bioanalyzer; Agilent Technologies, Santa Clara, CA) coding 3’ polyadenylated messenger RNA (mRNA) was extracted from lungs of E18.5 mice (n = 7) and purified with the Dynabeads mRNA Purification Kit (Invitrogen, Carlsbad, CA). Ambion RNA Fragmentation Reagents (Ambion, Inc., Foster City, CA) were used to fragment mRNA. Double-stranded complementary DNA (cDNA) was made with the use of the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) and random hexamer primers (Invitrogen; 50 ng/µL). DNA sequencing libraries were generated with cDNA according to manufacturer’s protocol. Cluster generation and sequencing was performed on the Illumina cBot station and Illumina Hiseq 2000 (Illumina, San Diego, CA) producing 114,868,849 60bp single end reads (Supplemental Table 1; Supplemental Figure 1 ). Reads were aligned to the mouse genome assembly (NCBI37/UCSC mm9) with Tophat2,18 (link) and differentially expressed genes were identified with the intersection of Cuffdiff19 ,20 (link) and NOISeq.21 (link) Programs were run with standard settings and corrected for multiple testing with false discovery rate. Gene pathway interactions were determined with Ingenuity Pathway Analysis (IPA) software (QIAGEN, Redwood City, CA) and DAVID Gene Ontology.22 (link)
Ambion rna fragmentation reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ambion RNA Fragmentation Reagents are a set of solutions used to fragment RNA samples in a controlled manner. The reagents enable consistent and reproducible fragmentation of RNA, which is a necessary step in various RNA-related applications such as library preparation for RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (3 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Cbot station, by Illumina (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Epimark n6 methyladenosine enrichment kit, by New England Biolabs (1 mentions)
Library quant kit for, by Illumina (1 mentions)
Nebnext ultra directional rna library preparation kit for, by Illumina (1 mentions)
Rna clean and concentrator 5, by Zymo Research (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Protein a magnetic bead, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rabbit anti m6a antibody, by Synaptic Systems (1 mentions)
Ribominus eukaryote system v2, by Thermo Fisher Scientific (1 mentions)
Anti m6a polyclonal antibody, by Synaptic Systems (1 mentions)
Protein a bead, by Thermo Fisher Scientific (1 mentions)
6 protocols using ambion rna fragmentation reagent
1
RNA-seq analysis of E18.5 mouse lung transcriptome
2
m6A-seq Library Preparation and Sequencing
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Total RNA was extracted using the Qiagen RNeasy Mini kit (catalog no./ID: 74104). The following protocol was performed with 35 to 50 μg of total RNA. mRNA was purified with Dynabeads mRNA purification kit (Invitrogen Catalog No. 61006). Fragmentation of the mRNA was performed using the Ambion RNA fragmentation reagents (catalog no. AM8740), and then the mRNA was subjected to the RNA Clean and Concentrator-5 (catalog no. R1013 from Zymo Research). mRNA quality was checked by Aligent BioAnalyzer 2100 using the RNA 6000 Pico Kit (part number 5067-1513). One round of m6A immunoprecipitation was performed using the EpiMark N6-Methyladenosine Enrichment Kit (NEB no. E1610S) and protocol. m6A-seq libraries were prepared using the NEBNext Ultra Directional RNA library preparation kit for Illumina (NEB no. E7760). Library quality was checked by Agilent BioAnalyzer 2100 using the High Sensitivity DNA kit (part number 5067-4626), and libraries were quantified using the Library Quant Kit for Illumina (NEB no. 7630). Libraries were then sequenced using a NextSeq 500 platform [75–base pair (bp) single-end reads]. All kits were used following the manufacturer’s instructions.
3
Nuclear m6A Immunoprecipitation and RNA Analysis
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The nuclear fraction from 4 × 107 cells was isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). RNA was extracted from the nuclear fraction using TRIzol (Invitrogen) and then fragmented into sizes between 60 and 200 nucleotides in length using Ambion® RNA Fragmentation Reagents (Thermo Scientific). The fragmented RNA was precipitated by adding one-tenth volumes of 3 M sodium acetate (pH 5.2) and glycogen (100 μg/mL final) and 2.5 volumes of 100% ethanol. Rabbit anti-m6A antibody (Synaptic Systems, Germany) and rabbit normal IgG control bound to protein A magnetic beads (Millipore) were used for immunoprecipitation. The immunoprecipitated RNA was extracted using phenol:chloroform:isoamyl alcohol. Then, the aqueous solution was precipitated and resuspended in 10 μL of RNase-free water. The RNA was analyzed by qRT-PCR.
4
High-Throughput PEDV m6A Methylation Analysis
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High-throughput sequencing of PEDV methylation was carried out by m6A-seq following a previously described protocol (Dominissini et al., 2013 (link)). In brief, rRNA was removed from the total RNA by the RiboMinus Eukaryote System v2 (Thermo, USA). Then, the RNA was fragmented using Ambion RNA Fragmentation Reagents (Thermo, USA) and mixed with 25 μg of affinity purified anti-m6A polyclonal antibody (Synaptic Systems, Germany) at 4 °C for 2 h. Sequencing libraries were prepared with eluted RNA, as well as input RNA, using the TruSeq RNA sequencing (RNA-seq) kit (Illumina, USA). Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer's instructions and was performed by Novel Bioinformatics Company.
5
m6A-IP RNA Sequencing Protocol
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m6A-IP and library preparation were carried out according to the reported procedure45 (link). In brief, poly-A-purified RNA was fragmented with Ambion RNA Fragmentation reagent (Ambion, Carlsbad, CA, USA), and incubated with m6A primary antibody at 4°C for 2 h. The mixture was immunoprecipitated through incubation with Protein A beads (Thermo Fisher, MA, USA) at 4°C for 2 h. Followed by washing for three times, the Captured RNA was eluted with m6A nucleotide solution and purified with RNA Clean and Concentrator Kit (Zymo, LA, USA). Sequencing was performed on Illumina HiSeq 2000 according to the manufacturer’s recommendations.
6
m6A-RNA Immunoprecipitation and Quantification
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mRNA was prepared as described above, and fragmented using Ambion RNA Fragmentation reagent (Ambion, Carlsbad, CA, USA) at 70°C for 15 min. A small portion (10%) of the RNA fragments was collected to be used as input sample. MeRIP-qPCR was performed according to previously protocol9 (link). In brief, fragmented mRNA was incubated immunoprecipitated with anti-m6A antibody (Synaptic Systems) in immunoprecipitation buffer (RNase inhibitor, 50 mm Tris-HCl, 750 mm NaCl and 0.5% (vol/vol) Igepal CA-630 (Sigma, St. Louis, MO, USA)) at 4 °C for 2 h with rotation. The m6A antibody-RNA mixture was incubated with Dynabeads protein A (Invitrogen, CA, USA) at 4°C for 2 h with rotation. The bound RNA was eluted twice by competition with M6A 5’-monophosphate sodium salt (Sigma, St. Louis, MO, USA) at 4°C for 1 h. Following ethanol precipitation, the input RNA and immunoprecipitated m6A RNAs were reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen, CA, USA). m6A enrichment was determined by qPCR analysis. The primers used for MeRIP-qPCR were listed in Table S2 .
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