title: GSM4875673: m6A-RP.Mettl3-WT.IP.rep1; Mus musculus; RIP-Seq
library_construction_protocol: total RNA RIP assay was performed based on GoldCLIP33 with modifications. The cells expressing Halo-GFP or Halo-YTHDC1 were harvested, lysed, and pulled-down using Halo-tagged beads (Promega, #G7282) as previously described33, except that no nuclease digestion or RNA fragmentation conducted throughout the experiment. The binding efficiency of Halo-tagged protein to the beads was evaluated by western blot. After the denaturing wash, the RNAs were released from the proteins by PK enzyme (NEB, #P8107S) digestion. For RIP-RT-qPCR, the equivalent input and IP RNAs were reverse-transcribed by SuperScript III Reverse Transcriptase (Invitrogen, #18080044) using random hexamers, followed by qPCR to quantify the transcripts. The enrichment score of a transcript was the ratio of the quantified IP fraction to its total input, using GFP as a standard for normalization. For RIP-seq, 200-500 ng RNAs from the input and IP samples were used to generate the libraries using VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (VAHTS, #NR603) according to the manufacturer's instructions.