The required amount of DNase I–treated total RNA (File S1A ) was removed from storage at −80° and diluted to 9 μl with DNase/RNase-free water. Then 1 μl of 10× RNA fragmentation buffer (AM8740, Ambion) was added, and the sample was mixed and incubated for 15 min at 70°. The fragmentation reaction was terminated by adding 1 μl of stop solution (AM8740, Ambion), and the mixture was placed on ice until use. Next, Dynabeads Oligo(dT)25 (75 µg of beads; 61002, Ambion) were washed and prepared according to the manufacturer’s instructions. The fragmented total RNA was heated to 65° for 2 min to disrupt secondary structures, immediately placed on ice, added to the washed Dynabeads, and mixed thoroughly. The mixture then was rotated continuously for 5 min at room temperature to allow binding of the poly(A)+ RNA to the beads. Bead-bound poly(A)+ RNA was eluted with 10 µl of elution buffer (10 mM Tris-HCl, pH 7.5) as directed. The sample was incubated with Dynabeads an additional 5 min and eluted as described earlier to further enrich poly(A)+ RNA. The concentration of fragmented poly(A)+ RNA was measured with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE).
Am8740
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AM8740 is a centrifuge designed for laboratory use. It has a maximum speed of 14,000 RPM and a maximum RCF of 20,000 xg. The centrifuge can accommodate 6-12 test tubes or microcentrifuge tubes, depending on the rotor configuration.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads oligo dt 25, by Thermo Fisher Scientific (3 mentions)
Agilent scanner, by Agilent Technologies (2 mentions)
Dynabeads oligo t magnetic beads, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
A63880, by Beckman Coulter (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ab151230, by Abcam (2 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Protein a g bead, by Thermo Fisher Scientific (2 mentions)
Dna free kit, by Thermo Fisher Scientific (2 mentions)
Clean up columns, by Zymo Research (2 mentions)
Ab208577, by Abcam (2 mentions)
Stranded rnaseq kit, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Anti m6a antibody, by Synaptic Systems (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rna fragmentation buffer, by Thermo Fisher Scientific (1 mentions)
M3148, by Merck Group (1 mentions)
Rneasy minelute cleanup kit, by Thermo Fisher Scientific (1 mentions)
20 protocols using am8740
1
Fragmentation and Isolation of Poly(A)+ RNA
2
RNA Isolation and Fragmentation Protocol
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For each experiment, approximately 100 μg of total RNA was isolated from cells with TRIzol according to the manufacturer’s instructions. Approximately 10 μg PolyA RNA was isolated using Magnosphere Ultrapure mRNA Purification Kit (Takara) according to the manufacturer’s instructions. PolyA RNA was ethanol precipitated with 2.5 M ammonium acetate and 70% ethanol in a solution containing 50 μg/ml GlycoBlue Co-precipitant (AM9515, Invitrogen). RNA was resuspended in 10 μl and fragmented with 10× fragmentation buffer (AM8740, Invitrogen) at 75°C for 8 minutes and immediately quenched with 10× Stop Reagent (AM8740, Invitrogen) and placed on ice to generate fragments 30 to 150 nucleotides in length.
3
Fluorescent Labeling and Hybridization of RNA
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RNA was labelled using the Kreatech ULS™ Fluorescent Labeling Kit for Agilent arrays (Kreatech EA-023) as described previously [8 (link)]. The labelled RNA was fragmented by adding 2 μl 10× fragmentation buffer, incubating for 15 min at 70 °C, then adding 2 μl stop solution (Ambion® AM8740). Labelled RNA (20 μl) was added to 27.5 μl Kreatech blocking reagent (Kreatech EA-023), 55 μl of 2× Hybridisation buffer and 7.5 μl of molecular grade water. Arrays were hybridised overnight at 65 °C, then washed in Gene Expression wash buffer 1 (Agilent 5188-5327) for 1 min at room temperature with agitation, then in Gene Expression wash buffer 2 for 1 min at 37 °C with agitation. Slides were scanned immediately using an Agilent Scanner.
4
Nascent RNA sequencing by Bru-seq
5
Quantifying mRNA m6A Methylation by MeRIP-qPCR
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MeRIP-qPCR was performed according to a previously reported method69 (link). Briefly, Total RNA was extracted from H9C2 cardiomyocytes by miRNeasy Mini Kit (QIAGEN, Cat# 217004) with DNase I on column treatment (QIAGEN, Cat# 79254). mRNA was isolated from total RNA via oligo(dT) polystyrene beads (Sigma, Cat#MRN10) and fragmented (Ambion, AM8740) at 70 °C for 15 min. 1 μg of fragmented mRNA was saved as input sample. The remaining fragmented mRNA was immunoprecipitated with an anti-m6A antibody (Synaptic Systems, Cat#202003) coupled Dynabeads Protein G (Invitrogen, Cat#10004D). PCR primers of specific genes were designed based on the meRIP-seq data and RMBase database which integrated the public epitranscriptome sequencing data and annotated the RNA m6A methylation sites of genes33 (link). m6A enrichment was determined by qPCR with specific gene primers listed in Supplementary Table 3 .
6
WTTS-Seq: Comprehensive RNA-Seq Protocol
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In the present study, we used our WTTS-Seq protocol23 (link),81 (link) to construct libraries for all rats. For each individual sample, 2.5 μg of total RNA was chemically fragmented using RNA fragmentation buffer (AM8740, Ambion) as the first step, then enrichment of poly(A) + RNA was accomplished with Dynabeads oligo(T) magnetic beads (61002, Ambion) followed by reverse transcription with SuperScript III Reverse Transcriptase (18080, Invitrogen) to synthesize first strand cDNA. Next, both 5′- and 3′- adaptors were added to fit the Ion Torrent sequencing platform. All RNA molecules were removed by both RNases H (M0297L, NEB) and I (EN0601, Thermo Scientific) to prevent contamination, 250–500 bp first-strand cDNA molecules were selected with solid-phase reversible immobilization beads (A63880, Beckman Coulter), and second-strand cDNA was synthesized by PCR. Lastly, all libraries were sequenced using an Ion PGM™ Sequencer at Washington State University.
7
Total RNA Extraction and Fragmentation
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Washed cell pellets were prepared as described in “DNA sequencing”. Total RNA extraction was performed using the RNeasy MinElute Cleanup kit (Qiagen #74204). First, 600 μl of RLT buffer with 1% β-mercaptoethanol (Sigma-Aldrich #M3148) and 200 mg of glass beads were added to the pellet, and the cells were lysed on an IKA Vibrax VXR for 20 min at 4 °C with subsequent centrifugation at 17,000 × g for 2 min. The supernatant was transferred to a new tube and mixed with an equal volume of 70% ethanol (Pharmco #111000200), and transferred to an RNeasy column to proceed with RNA extraction using the manufacturer’s protocol. RNA was eluted from the column with 22 μl of nuclease-free water (Fisher #BP2819). Total RNA was subsequently fragmented using RNA fragmentation reagents (Ambion #AM8740) and cleaned up with the RNeasy MinElute Cleanup kit. RNA was eluted from the column with 9 μl of nuclease-free water.
8
RNA Labeling and Microarray Hybridization
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RNA was labelled using the Kreatech ULS™ Fluorescent Labeling Kit for Agilent arrays (Kreatech EA-023). The labelled RNA was fragmented by adding 2 μl 10x fragmentation buffer, incubating for 15 minutes at 70°C, then adding 2 μl stop solution (Ambion® AM8740). Labelled RNA (20 μl) was added to 27.5 μl Kreatech blocking reagent (Kreatech EA-023), 55 μl of 2x Hybridisation buffer and 7.5 μl of molecular grade water. Arrays were hybridised overnight at 65°C, then washed in Gene Expression wash buffer 1 (Agilent 5188–5327) for 1 minute at room temperature with agitation, then in Gene Expression wash buffer 2 for 1 minute at 37°C with agitation. Slides were scanned immediately using an Agilent Scanner.
9
RNA-seq Protocol for Muscle Tissue
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In the present study, two male KO and two male WT mice were used per gene model. For each individual, 2.5 μg of total RNA derived from muscle were chemically fragmented with RNA fragmentation buffer (AM8740, Ambion). After that, poly(A)+ RNA was enriched by Dynabeads oligo(T) magnetic beads (61002, Ambion). Reverse transcription with SuperScript III Reverse Transcriptase (18080, Invitrogen) was used to synthesize first cDNA strand with integration of both 5′adaptor and 3′adaptor. After all RNA molecules were removed by both RNases H (M0297L, NEB) and I (EN0601, Thermo Scientific), solid-phase reversible immobilization beads were used to select 300–600 bp first-strand cDNA molecules (A63880, Beckman Coulter), and second-strand cDNA was synthesized by PCR. Size selection was repeated as described above and the final libraries were sequenced using an Ion PGM™ system at Washington State University.
10
m6A-Seq Library Preparation Protocol
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Polyadenylated RNAs (polyA+ RNAs) were prepared using Dynabeads mRNA DIRECT kit (ambion).
PolyA+ RNAs were then fragmented using fragmentation buffer (Ambion #AM8740) to yield ~200 nt lengths RNA fragments. Polyclonal anti-m6A antibody (Synaptic Systems) was incubated with fragmented RNAs in RIP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP40) supplemented with RNase inhibitors, RNasin (promega) and Superase-in (Ambion), for 4 hours at 4 °C. Along with this, Dynabeads protein A (Invitrogen #10002D) was washed and pre-blocked with 0.5 mg/ml BSA for 2 hours at 4 °C. Beads were then added to antibody-RNA complex and incubated for an additional 1 hour at 4 °C. Bound RNA was eluted after extensive washing with RIP buffer by competitive elution with buffer containing 7 mM m6A (Abcam #ab145715) twice each for 1 hour at 4 °C. Eluted RNAs were then precipitated with ethanol and used for m6A-sequencing library preparation.
Sequencing libraries were prepared using SMARTer Stranded RNA-seq kit (Clontech #634837) according to the manufacturer's protocol. Purified m6A-seq libraries were sequenced on an Illumina MiSeq platform with 10-20% PhiX control library (Illumina). The data is deposited in the Gene Expression Omnibus (GEO) under the accession numbers GSEXXXXXX.
PolyA+ RNAs were then fragmented using fragmentation buffer (Ambion #AM8740) to yield ~200 nt lengths RNA fragments. Polyclonal anti-m6A antibody (Synaptic Systems) was incubated with fragmented RNAs in RIP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP40) supplemented with RNase inhibitors, RNasin (promega) and Superase-in (Ambion), for 4 hours at 4 °C. Along with this, Dynabeads protein A (Invitrogen #10002D) was washed and pre-blocked with 0.5 mg/ml BSA for 2 hours at 4 °C. Beads were then added to antibody-RNA complex and incubated for an additional 1 hour at 4 °C. Bound RNA was eluted after extensive washing with RIP buffer by competitive elution with buffer containing 7 mM m6A (Abcam #ab145715) twice each for 1 hour at 4 °C. Eluted RNAs were then precipitated with ethanol and used for m6A-sequencing library preparation.
Sequencing libraries were prepared using SMARTer Stranded RNA-seq kit (Clontech #634837) according to the manufacturer's protocol. Purified m6A-seq libraries were sequenced on an Illumina MiSeq platform with 10-20% PhiX control library (Illumina). The data is deposited in the Gene Expression Omnibus (GEO) under the accession numbers GSEXXXXXX.
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