RNA was fragmented by an RNA fragmentation reagents kit (Thermo Fisher) at 95 °C for 10 mins and then purified by 10 μl 3-M NaOAc (pH 5.2), 24 μl 5-μg/μl glycogen, and 372 μl 96% cold ethanol. For facilitating MgR binding of fragments with complex secondary structures after solubilization in water, every 1 μg RNA solution was ligated with 3 μl 25-μM poly(A)-ssRNA adaptor (pAGCUAAAAAAAAAAAAp, synthesized by GenScript Biotech Co.) at 16 °C overnight. The products were then reacted with 30 μg MgR in a 60 μl final volume with 20 mM Tris-Cl (pH 8.0), 100 mM KCl, and 0.01 mM ZnCl2 at 37 °C for 30 min. Next, the products were purified by an RNA Clean & Concentration-5 kit (Zymo Research), followed by 5′ phosphorylation (39 μl RNA, 5 μl (10X T4 PNK Reaction Buffer), 5 μl ATP (10 mM), 1 μl T4 PNK (3′ phosphatase minus, New England BioLabs)). Finally, we constructed the NGS library using the products from the last step following the NEBNext Small RNA Library Prep Set for Illumina (New England BioLabs) protocol. Fragments smaller than 50 nt were extracted from a 6% PAGE gel and sent for NGS using Illumina X-Ten. The exact process prepared the NC sample (negative control group) without MgR protein.
Rna fragmentation reagents kit
Manufactured by Thermo Fisher Scientific
The RNA fragmentation reagents kit is designed to fragment RNA samples for downstream applications such as next-generation sequencing. The kit contains reagents necessary to prepare fragmented RNA samples.
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Lab products found in correlation
2 protocols using rna fragmentation reagents kit
1
Preparing RNA-seq library from fragmented RNA
2
Quantifying m6A RNA modifications
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MeRIP-RT-qPCR was performed as previously described with minor modifications [21 (link)]. Briefly, the total RNA was fragmented with the RNA Fragmentation Reagents Kit (AM8740, Thermo Fisher) according to the manufacturer’s instructions. The sizes of RNA fragments were verified to be around 100 nucleotides using an Agilent 2100 Bioanalyzer System. Fragmented RNA at 6 μg were added to 10 μl Slurry of PierceTM Protein A Agarose beads pre-incubated with 1 μg of an anti-m6A polyclonal antibody (202-003, Synaptic Systems) by rocking at 4 ˚C for 3 h. The beads were washed 7 times with ice cold PBS. The beads were incubated with elution buffer at pH 7.4 containing 200 mM Tris–HCl, 1 M NaCl, 1% Igepal CA-630, and 20 mM N6-methyladenosine 5′-monophosphate sodium salt for 1 h at 4 ˚C. Eluate was collected and subjected to GeneJET RNA Cleanup and Concentration Micro Kit for RNA purification (K0841, Thermo Fisher). Total RNA at 1 μg was used as the input. All eluted RNA were used for reverse transcription.
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