Polyattract mrna purification kit

The TRIzol Reagent was purchased from Invitrogen (Carlsbad, CA, USA). The PolyATtract mRNA purification kit was bought from Promega (Madison, WI, USA). The Taq DNA polymerase and the restriction enzymes Nde I and Xho I were obtained from Takara Biomedical Technology (Beijing, China). The T4 DNA Ligase was bought from New England Biolabs (Beijing, China). The plasmid extraction and DNA gel extraction kits were purchased from BioTeke (Beijing, China). The tryptone, yeast extract, NaCl and other salts, agar and Tris base were purchased from Merck (Darmstadt, Germany). The IPTG and kanamycin were obtained from Yeasen Biotech (Hongkong, China). The EZ-Link NHS-Biotin reagents were from Thermo Scientific (Waltham, MA, USA).

Cells were collected, and total RNA was extracted with TRIzol reagent. mRNA was purified with a PolyATtract mRNA Purification Kit (Promega) and fragmented with a MeRIP Kit (Millipore). Magnetic beads were incubated with 20 μl of protein G and an anti-m⁵C antibody (Abcam) at 4 °C for 4 h. Then, the anti-m⁵C antibody-coated protein G magnetic beads were added to the fragmented mRNA, and the mixture was incubated at 4 °C overnight. After washing with buffer solution (0.02% Triton X-100, 0.1% BSA) 3 times, the coprecipitated RNA was isolated with TRIzol reagent, and the target RNA expression was quantified via RT‒qPCR.