- Home
- Protocols
-
Ask a
question
A 1–3 μg total RNA and m6A spike-in control mixture was added to 300 μL 1IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP40, 40 U/μL RNase Inhibitor) containing 2 μg anti-m6A rabbit polyclonal antibody (Synaptic Systems). A 20 L Dynabeads™ M-280 Sheep Anti-Rabbit IgG suspension per sample was blocked with freshly prepared 0.5% BSA at 4 °C for 2 h, washed three times with 300 μL 1IP buffer, and re-suspended in the total RNA-antibody mixture prepared above. The RNA binding to the m6A-antibody beads was carried out with head-over-tail rotation at 4 °C for 2 h. The enriched RNA was eluted with 200 μL Elution buffer at 50 °C for 1 h, after which the co-precipitated RNA was isolated and qRT-PCR detection was performed on the RNA as the previously described. Sequences of the primers are listed in Additional file 1: Table S1.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Post a Question
