GSM8625548: WT, replicate 1, IgG; Mus musculus; RIP-Seq
A total of 10 to 50 micrograms of RNA was subjected to immunoprecipitation using the GenSeq acRIP Kit (GenSeq Inc.) in accordance with the manufacturer's instructions. In summary, total RNA was randomly fragmented to a length of approximately 200 nt using RNA fragmentation reagents. The anti-ac4C antibody or control IgG (sc-2027, Santa Cruz) was conjugated to Protein A/G Dynabeads (Share-Bio, SB-PR001) in PBS and incubated for one hour at room temperature. Subsequently, the RNA fragments were incubated with the bead-conjugated antibodies, which were rotated at 4°C for four hours. Subsequently, the RNA/antibody complexes were subjected to multiple rounds of washing in order to remove any unbound materials. The captured RNA was subsequently eluted from the complexes and purified to ensure its suitability for subsequent analysis. Ribosomal RNA (rRNA) depletion was performed on the RNA samples using the NEBNext rRNA Depletion Kit (New England Biolabs, Inc.). RNA libraries for immunoprecipitation (IP) and input samples were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc.) in accordance with the standard protocol. The quality of the libraries was evaluated using the Agilent 2100 Bioanalyzer (Agilent), and sequencing was conducted on the NovaSeq platform (Illumina).