GSM5322385: METTL8-His6-2xFLAG; Homo sapiens; OTHER
Crosslinked RNA-protein complexes were enriched on anti-FLAG beads (Sigma) and eluted using 3x FLAG peptide. RNAs associated with His6-2xFLAG-tagged proteins were subjected to partial RNase digestion using 1 U RNace-It ribonuclease cocktail for 3 min at 37°C. Complexes were immobilized on NiNTA under denaturing conditions (6 M guanidium hydrochloride). Illumina TruSeq RA3 and RA5 adapters were ligated to the 3' and 5' ends of the RNA fragments enriched on the matrix. Complexes were eluted using immidazole and precipitated using trichloroacetic acid. Protein-RNA complexes were separated by NuPAGE and transfered to a nitrocellulose membrane. Relevant regions of the membrane containing complexes of interest were excised and RNAs eluted by Proteinase K treatment. RNAs were isolated, and reverse transcribed using Superscript III reverse transcriptase to produce a cDNA library that was amplified by PCR (Haag et al., 2017).