BrdU-CLIP cDNA Library Preparation

The eluted RNA from the m⁶A-IP step and the input RNA fragments were first treated with T4 PNK to remove its 3′ phospho group and then ligated to 68 pmol preadenylated DNA linker (L32N from IDT) with T4 RNA ligase 2 and truncated KQ (New England Biolabs, catalog no. M0373L) overnight at 16°C. This ligation mixture was subject to 8% PAGE purification to harvest the ligated product. The purified RNAs from m⁶A-IP and m⁶A-CLIP and their input RNA fragments were all subjected to the same BrdU-CLIP cDNA library preparation. The detailed BrdU-CLIP cDNA library protocol was described previously (Weyn-Vanhentenryck et al. 2014 (link)), and we practiced a similar procedure with an improved RT primer set that starts with three “D” (no C) nucleotides on the 5′ end and improves ligation efficiency.